Renal cell populations and uses thereof

Abstract

The present invention relates to an enriched heterogeneous mammalian kidney cell population characterized by a biomarker, and a method for producing and using the same.

Description

Technology Field

[0001] Cross-reference of related applications

[0002] This application claims priority to U.S. provisional application No. 61 / 718,150, filed on October 24, 2012, titled “Isolated Regenerative Renal Cells and Uses Thereof,” and U.S. provisional application No. 61 / 876,616, filed on September 11, 2013, titled “Renal Cell Populations and Uses Thereof.”

[0003] Technology field

[0004] The present disclosure generally relates to an enriched heterogeneous mammalian kidney-derived cell population, and provides in this specification a method for identifying the cell population, a method for their use in the manufacture of regenerative medicine therapies, and a method for treating kidney disease by administering the enriched heterogeneous kidney-derived cell population to a mammalian subject. Background Technology

[0005] Background of the Invention

[0006] Collagen and gelatin-based biomaterials have been successfully used for various tissue manipulation applications (Rohanizadeh et al . J Mater Sci Mater Med 2008; 19: 1173-1182; Takemoto et al . Tissue Eng Part A 2008; 14: 1629-1638; Young et al. J Control Release 2005; 109: 256-274). Both of these macromolecules are characterized by excellent biocompatibility and low antigenicity (Cenni et al. J Biomater Sci Polym Ed 2000; 11: 685-699; Lee et al. Int J Pharm 2001; 221: 1-22; Waksman et al. J Immunol 1949; 63: 427-433); however, since gelatin is obtained by the hydrolysis of collagen, it has the following specific advantages over the latter: (a) it is easily available and easy to use; (b) it offers options regarding molecular weight and bloom (i.e., control over physical properties); and (c) it is more flexible regarding chemical modification and easier to manufacture. Furthermore, from a biological perspective, gelatin maintains cell adhesion properties and cytocompatibility similar to collagen (Engvall). et al . Int J Cancer 1977; 20: 1-5; Kim et al . Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108: e94-100).

[0007] Various methods have been reported for the crosslinking of these macromolecules to extend their in vivo retention (in tissue manipulation applications), delay their biodegradation, or tune their drug release capabilities (when used as drug carriers). Numerous methods have been disclosed for the chemical or photochemical crosslinking of collagen or gelatin (Adhirajan et al . J Microencapsul 2007; 24:647-659; Chang et al . Macromol Biosci 2007; 7: 500-507; Gagnieu et al . Biomed Mater Eng 2007; 17:9-18; Kimura et al. J Biomater Sci Polym Ed 2010; 21: 463-476; Ma et al . J Biomed Mater Res A 2004; 71: 334-342; Vandelli et al . Int J Pharm 2001; 215: 175-184; Vandelli et al Many of these procedures aim to reduce the sensitivity of these biomaterials to enzymatic degradation and extend their in vivo retention time (Chang et al . supra 2007; Ma et al . supra (2004). Other crosslinking methods are typically used to obtain gelatin or collagen-based biomaterials suitable as carriers for sustained-release drugs, proteins, or nucleic acids (Kimura supra 2010; Vandelli supra 2004; Kommareddy et al . Nanomedicine 2007; 3: 32-42; Sehgal et al . Expert Opin Drug Deliv 2009; 6: 687-695; Sutter et al J Control Release 2007; 119: 301-312). A class of crosslinking agents widely used for collagen, gelatin, and other tissue manipulation-compatible systems is carbodiimide (Adhirajan supra 2007; Olde Damink et al . Biomaterials 1996; 17: 765-773; Pieper et al . Biomaterials 2000; 21: 581-593; Cornwell et al. Clin Podiatr Med Surg 2009; 26: 507-523). These molecules are known as zero-length crosslinkers and act by mediating the formation of amide bonds between the carboxyl and primary amine functionalities present in the species to be crosslinked. Additionally, carbodiimides are less cytotoxic compared to other common crosslinking agents (e.g., glutaraldehyde) (Lai et al . J Mater Sci Mater Med 2010; 21: 1899-1911). Glutaraldehyde is used as a crosslinking agent in Cultispher™ beads. Burg U.S. Patent No. 6,991,652 describes a tissue manipulation composite containing a three-dimensional support structure for cells that may be delivered to a subject.

[0008] Regenerative medicine technology provides next-generation therapeutic options for chronic kidney disease (CKD). Presnell et al WO / 2010 / 056328 and Ilagan et al PCT / US2011 / 036347 describes isolated bioactive renal cells, including tubular and erythropoietin (EPO)-producing renal cell populations, a method for isolating and culturing them, and a method for treating a target in need of them with the cell populations.

[0009] In tissue manipulation and regenerative medicine applications, there is a need for therapeutic agents suitable for delivering activators, such as biologically active cells, to targets that require them.

[0010] The kidneys are complex organs that perform many functions to keep the blood clean and chemically balanced. In addition to removing waste products, the kidneys release the following three important hormones:

[0011] Erythropoietin, or EPO, stimulates the bone marrow to produce red blood cells.

[0012] Renin regulates blood pressure; and

[0013] Calcitriol, the active form of vitamin D, helps maintain calcium for normal chemical balance in the body and for bones.

[0014] To perform these functions, the kidney contains numerous different cell types. However, not all cells require a regenerative response, and identifying combinations of cells useful for inducing a regenerative response has become a subject of research. Therefore, there remains a need for a method to identify heterogeneous kidney cell populations, namely bioactive cells, for use in the therapeutic agents disclosed herein.

[0015] Summary of the Invention

[0016] A heterogeneous population of mammalian kidney cells from mammalian kidney tissue is disclosed herein. A method for isolating and purifying a mammalian kidney-derived cell population is provided. The unique population of mammalian kidney-derived cells is characterized by phenotypic features, for example, a biomarker phenotype. The biomarker expression phenotype is retained after multiple passages of the mammalian kidney-derived cell population in culture and is suitable for use in the preparation of regenerative therapies.

[0017] Selection populations of human kidney cell populations characterized by specific biomarkers and their uses are described in this specification.

[0018] Selected human kidney cell populations allow for the use of a smaller number of cells to provide regenerative stimulation. This smaller number is beneficial because it lowers the likelihood of negative immunological events and provides regenerative stimulation. Selected kidney cell populations do not require a large proportion of stem cells, which must be effective as regenerative stimuli. Selected kidney cell populations can be recovered from diseased kidneys.

[0019] In one aspect, a method for identifying and / or characterizing a heterogeneous renal cell population is provided. In one embodiment, the heterogeneous renal cell population is characterized by the phenotypic expression of a biomarker. In a specific embodiment, renal cells are identified by one or more reagents that allow detection of the biomarker on / in the heterogeneous renal cell population. Detection of the biomarker may be performed by any suitable method, e.g., those based on immunofluorescence microscopy, flow cytometry, optical-fiber scanning cytometry, or laser scanning cytometry. In one embodiment, a method for identifying a heterogeneous renal cell population suitable for inducing a transplantation and / or regenerative response comprises the following steps:

[0020] Step of isolating cells from a mammalian kidney sample;

[0021] A step of exposing the isolated cells to one or more labeled detection moiety, wherein each labeled detection moiety targets a different biomarker and is labeled with a different label;

[0022] A step of determining the percentage of cells expressing each of the above biomarkers.

[0023] In one specific example, the cell population is Tables 12.2 and 12.3 It is a population of SRC cells expressing two or more biomarkers listed in ). In one embodiment, the biomarker is Table 12.4 It has expression levels as provided in. In a specific example, the SRC cell population has expression levels greater than 18% and 80% for GGT-1 and CK18, respectively.

[0024] In one aspect, an injectable, therapeutic formulation containing an activator, such as a bioactive cell, is provided. In one embodiment, the injectable formulation comprises a bioactive cell and a temperature-sensitive cell-stabilizing biomaterial. In another embodiment, the temperature-sensitive cell-stabilizing biomaterial maintains (i) a substantially solid state at 8°C or lower and / or (ii) a substantially liquid state at ambient temperature or higher. In one other embodiment, the bioactive cell comprises kidney cells as described herein. In another embodiment, the bioactive cell is substantially uniformly dispersed throughout the entire volume of the cell-stabilizing biomaterial. In another embodiment, at about 8°C to about ambient temperature, the biomaterial is in a solid-to-liquid transition state. In one embodiment, the substantially solid state is a gel state. In another embodiment, the cell-stabilizing biomaterial comprises a hydrogel. In one other embodiment, the hydrogel comprises gelatin. In another embodiment, gelatin is present in a formulation of about 0.5% to about 1% (w / v). In one embodiment, gelatin is present in a formulation of about 0.75% (w / v). In yet another embodiment, the formulation further comprises a cell viability agent. In one other embodiment, the cell viability agent comprises an agent selected from the group consisting of antioxidants, oxygen carriers, immunomodulatory factors, cell recruitment factors, cell adhesion factors, anti-inflammatory agents, immunosuppressants, angiogenesis factors, and wound healing factors. In some embodiments, the cell viability agent is an antioxidant. In one embodiment, the antioxidant is 6-hydroxy-2,5,7,8-tetramethylcloman-2-carboxylic acid. In another embodiment, 6-hydroxy-2,5,7,8-tetramethylcloman-2-carboxylic acid is present in an amount of about 50 μM to about 150 μM. In one other embodiment, 6-hydroxy-2,5,7,8-tetramethylcloman-2-carboxylic acid is present at about 100 μM.In some embodiments, the cell viability agent is an oxygen carrier. In one embodiment, the oxygen carrier is a perfluorocarbon. In another embodiment, the cell viability agent is an immunomodulator. In one embodiment, the cell viability agent is an immunosuppressant.

[0025] In another aspect, an injectable therapeutic formulation containing bioactive kidney cells is provided. In one embodiment, the formulation comprises bioactive kidney cells, about 0.75% (w / v) gelatin, and about 100 μM 6-hydroxy-2,5,7,8-tetramethylcloman-2-carboxylic acid, wherein the formulation is (i) substantially solid at about 8°C or lower, and (ii) substantially liquid at ambient temperature or higher. In another embodiment, the bioactive kidney cells are substantially uniformly dispersed throughout the entire volume of the cell-stabilizing biomaterial. In one other embodiment, the biomaterial is in a solid-to-liquid transition state at about 8°C to about ambient temperature. In another embodiment, the substantially solid state is a gel state. In some embodiments, the formulation further comprises a cell viability agent. In another embodiment, the cell viability agent comprises an agent selected from the group consisting of antioxidants, oxygen carriers, immunomodulatory factors, cell recruitment factors, cell adhesion factors, anti-inflammatory agents, angiogenic factors, and wound healing factors. In one embodiment, the cell viability agent is an oxygen carrier. In another embodiment, the oxygen carrier is a perfluorocarbon. In one other embodiment, the cell viability agent is an immunomodulator. In another embodiment, the cell viability agent is an immunosuppressant.

[0026] In one other aspect, the present disclosure provides a formulation described herein that further comprises biocompatible beads. In one embodiment, the biocompatible beads comprise a biomaterial. In another embodiment, the beads are cross-linked. In one other embodiment, the cross-linked beads have reduced sensitivity to enzymatic degradation compared to non-cross-linked biocompatible beads. In another embodiment, the cross-linked beads are carbodiimide-cross-linked beads. In one embodiment, the carbodiimide is selected from the group consisting of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), DCC-N,N'-dicyclohexylcarbodiimide (DCC), and N,N'-diisopropylcarbodiimide (DIPC). In another embodiment, the carbodiimide is 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). In one other embodiment, the cross-linked beads contain a reduced number of free primary amines compared to the non-cross-linked beads. In another embodiment, the number of free primary amines is detectable by spectrophotometry at about 355 nm. In some embodiments, the beads are inoculated into bioactive cells. In one embodiment, the bioactive cells are kidney cells. In another embodiment, the formulation further comprises additional biocompatible beads containing a temperature-sensitive biomaterial that maintains (i) a substantially solid state at ambient temperature and (ii) a substantially liquid state above 37°C. In one other embodiment, the biomaterial of the beads comprises a solid-to-liquid transition state between ambient temperature and about 37°C. In another embodiment, the substantially solid state is a gel state. In one embodiment, the biomaterial of the beads is a hydrogel. In another embodiment, the hydrogel contains gelatin. In one other embodiment, the beads contain gelatin in an amount of about 5% (w / v) to about 10% (w / v). In some embodiments, the additional biocompatible beads are spacer beads.In another embodiment, the spacer beads are not inoculated with bioactive cells.

[0027] In another aspect, the formulation of the present disclosure contains a product secreted by a renal cell population. In one embodiment, the formulation comprises a product secreted by a renal cell population and / or a bioactive cell. In one other embodiment, the bioactive cell is a renal cell. In another embodiment, the product comprises one or more of a paracrine factor, an endocrine factor, and a juxtacrine factor. In one other embodiment, the product comprises a vesicle. In another embodiment, the vesicle comprises a microvesicle. In one embodiment, the vesicle comprises an exosome. In another embodiment, the vesicle comprises a secreted product selected from the group consisting of a paracrine factor, an endocrine factor, a juxtacrine factor, and RNA. Brief explanation of the drawing

[0028] Fig. 1 silver This is a flowchart of the overall NKA manufacturing process described in this specification. Fig. 2 AD is a flowchart that provides additional details of the process illustrated in FIG. 1 and described in this specification. Fig. 3 This is a graph illustrating the rate of change during the culture period and the cell yield from 6 patients. Fig. 4 is 7% OptiPrep ® This is a figure of SRC banding in a density gradient. Reference is Example 5 It takes place in. Fig. 5 This is a bar graph plotting the expression of kidney cell markers in a human SRC population. Reference is Example 12 It takes place in. Fig. 6 This is a bar graph illustrating the enzyme activity of human SRC. Reference is Example 12 It takes place in. Figs. 7A-DIllustrated with NKA injection in the kidney: (a) a needle inserted into the renal cortex, (b) NKA delivery, (c) multiple delivery points in the kidney, and (d) the final injection of NKA (exemplary). Specific details for implementing the invention

[0029] Detailed description of the invention

[0030] The present specification discloses activators, such as therapeutic preparations for bioactive cells, methods for preparing the same, and methods for treating a subject in need of the preparation. Bioactive cell preparations may be suitable for heterogeneous mixtures or fractions of bioactive renal cells (BRCs). Bioactive renal cells may be isolated renal cells, including tubular and erythropoietin (EPO)-producing renal cells. BRC cell populations may comprise concentrated tubular and EPO-producing cell populations. BRCs may be derived from renal cell fractions from healthy individuals or may be themselves. Additionally, renal cell fractions obtained from unhealthy individuals are provided that may lack certain cellular components compared to the corresponding renal cell fractions of healthy individuals but still possess therapeutic properties. Furthermore, the present disclosure provides therapeutically active cell populations lacking cellular components compared to healthy individuals, wherein, in one embodiment, the cell population may be isolated and expanded from an autogenous source of various disease states.

[0031] Although bioactive cell formulations are described herein, the present disclosure also considers formulations containing various other activators. Other suitable activators include, but are not limited to, cell aggregates, cell-free biomaterials, products secreted from bioactive cells, large and small molecule therapeutic agents, and combinations thereof. For example, one type of bioactive cell may be combined with a biomaterial-based microcarrier with or without a therapeutic molecule, or another type of bioactive cell, which is a non-adherent cell, may be combined with a cell-free particle.

[0032] 1. Definition

[0033] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as generally understood by a person skilled in the art to which the present invention pertains. Principles of Tissue Engineering , 3 rd Ed. (Edited by R Lanza, R Langer, & J Vacanti), 2007 provides general guidance to those skilled in the art regarding many terms used in this application. Those skilled in the art will recognize many methods and materials similar or identical to those described herein that may be used in the practice of the invention. In fact, this specification is by no means limited to the methods and materials described.

[0034] The term “cell population” as used herein refers to a number of cells obtained by isolation from a suitable tissue source, primarily mammalian. The isolated cell population may subsequently be cultured in vitro. A person skilled in the art will recognize various methods for isolating and culturing a cell population for use in the present invention and various numbers of cells in a cell population suitable for use in the present invention. The cell population may be an undivided heterogeneous cell population derived from an organ or tissue, such as a kidney. For example, the heterogeneous cell population may be isolated from a tissue biopsy or whole organ tissue. Alternatively, the heterogeneous cell population may be derived from in vitro culture of mammalian cells established from a tissue biopsy or whole organ tissue. An undivided heterogeneous cell population may also be referred to as a non-enriched cell population. In one embodiment, the cell population contains bioactive cells.

[0035] The term "native organ" refers to an organ of a living subject. The subject may or may not be healthy. An unhealthy subject may have a disease associated with a specific organ.

[0036] The term "proper kidney" refers to the kidney of a living subject. The subject may or may not be healthy. An unhealthy subject may have kidney disease.

[0037] The term "regenerative effect" refers to an effect that provides benefit to a specific organ, such as the kidney. The effect may include, but is not limited to, a reduction in the degree of damage to the specific organ or the improvement, recovery, or stabilization of the specific organ's function. Kidney damage may take the form of fibrosis, inflammation, glomerular hypertrophy, etc., and may be associated with diseases related to the specific organ in the subject.

[0038] The term “mixture” as used herein refers to a combination of two or more isolated and concentrated cell populations derived from an undivided heterogeneous cell population. According to a specific embodiment, the cell population is a kidney cell population.

[0039] "Enriched" cell population or product refers to a cell population derived from an initial organelle cell population (e.g., an undivided heterogeneous cell population) that contains a specific cell type in a greater percentage than the percentage of cell types in the initial population. For example, an initial kidney cell population may be enriched for the first, second, third, fourth, fifth, etc. cell populations of interest. The terms "cell population," "cell product," and "cell prototype" as used herein are used interchangeably.

[0040] In one aspect, the term “enriched” cell population as used herein refers to an initial organ cell population (e.g., a cell suspension from a kidney biopsy or cultured mammalian kidney cells) containing a percentage of EPO-producing cells greater than the percentage of EPO-producing cells in the initial population. For example, the term “B4” is a cell population derived from an initial kidney cell population containing a greater percentage of EPO-producing cells, glomerular cells, and vascular cells compared to the initial cell population. A cell population may be enriched for one or more cell types and depleted for one or more other cell types. For example, in an enriched EPO-producing cell population, compared to a non-enriched cell population—that is, stromal fibroblasts and tubular cells in the initial cell population from which the enriched cell population originated—stromal fibroblasts may be enriched and tubular cells and collecting duct epithelial cells may be depleted. In all embodiments referring to EPO enrichment or the "B4" population, the enrichment cell population is a heterogeneous cell population containing cells capable of producing EPO in an oxygen-regulated manner, as evidenced by oxygen-regulated EPO expression from endogenous intrinsic EPO genes.

[0041] In another aspect, an enriched renal cell population containing a specific cell type, e.g., vascular, glomerular, or endocrine cells, in a greater percentage than the percentage of cell types in the initial cell population, may also lack or be deficient in one or more specific cell types, e.g., vascular, glomerular, or endocrine cells, compared to an initial renal cell population derived from a healthy individual or subject. For example, the term "B4'" or "B4 prime" refers, in one aspect, to cells derived from an initial renal cell population that lacks or is deficient in one or more cell types, e.g., vascular, glomerular, or endocrine cells, depending on the disease state of the source specimen, compared to a healthy individual. In one embodiment, the B4' cell population is derived from a subject having chronic kidney disease. In one embodiment, the B4' cell population is derived from a subject having focal segmental glomerulosclerosis (FSGS). In another embodiment, the B4' cell population is derived from a subject having autoimmune glomerulonephritis. In another aspect, B4' is a cell population derived from an initial cell population comprising all cell types, e.g., vascular, glomerular, or endocrine cells, to which one or more cell types, e.g., vascular, glomerular, or endocrine cells, are subsequently depleted or deficient. In another aspect, B4' is a cell population derived from an initial cell population comprising all cell types, e.g., vascular, glomerular, or endocrine cells, to which one or more specific cell types, e.g., vascular, glomerular, or endocrine cells, are subsequently enriched. For example, in one embodiment, the B4' cell population may be enriched with vascular cells but depleted with glomerular and / or endocrine cells. In another embodiment, the B4' cell population may be enriched with glomerular cells but depleted with vascular and / or endocrine cells.In another embodiment, the B4' cell population may be enriched with endocrine cells but depleted of vascular and / or glomerular cells. In another embodiment, the B4' cell population may be enriched with vascular and endocrine cells but depleted of glomerular cells. In a preferred embodiment, the B4' cell population possesses therapeutic properties either alone or in a mixed state with another enriched cell population, e.g., B2 and / or B3. For example, the B4' cell population is described herein in the Examples, e.g., Examples 11-13.

[0042] In another aspect, the enriched cell population may represent a cell population derived from the initial renal cell population discussed above, comprising a percentage of cells expressing one or more vascular, glomerular, and proximal tubular markers with some EPO-producing cells that is greater than the percentage of cells expressing one or more vascular, glomerular, and proximal tubular markers with some EPO-producing cells in the initial population. For example, the term "B3" represents a cell population derived from the initial renal cell population that contains a larger percentage of proximal tubular cells and vascular and glomerular cells compared to the initial population. In one embodiment, the B3 cell population contains a larger percentage of proximal tubular cells compared to the initial population but a smaller percentage of proximal tubular cells compared to the B2 cell population. In another embodiment, the B3 cell population contains a larger percentage of vascular and glomerular cell markers with some EPO-producing cells compared to the initial population, but contains a smaller percentage of vascular and glomerular cell markers with some EPO-producing cells compared to the B4 cell population.

[0043] In another aspect, the enriched cell population may also represent a cell population derived from the initial renal cell population discussed above, comprising a percentage of cells expressing one or more tubular cell markers that is greater than the percentage of cells expressing one or more tubular cell markers in the initial population. For example, the term "B2" refers to a cell population derived from the initial renal cell population that contains a larger percentage of tubular cells compared to the initial population. Additionally, the cell population enriched with cells expressing one or more tubular cell markers (or "B2") may contain some epithelial cells from the collecting duct system. Although the cell population enriched with cells expressing one or more tubular cell markers (or "B2") is relatively depleted of EPO-producing cells, glomerular cells, and vascular cells, the enriched population may contain these cells (EPO-producing, glomerular, and vascular) in smaller percentages compared to the initial cell population. Generally, one or more cell types are depleted such that the depleted cell population contains a smaller proportion of cell type(s) compared to the proportion of cell type(s) contained in the heterogeneous cell population before depletion. The cell types that may be depleted are any type of renal cell. For example, in a specific embodiment, the cell types that may be depleted include cells with a large particle size collecting duct and tubular system having a density of < about 1.045 g / ml, which is referred to as "B1". In another specific embodiment, the cell types that may be depleted include fragments and small cells with low particle size and viability having a density of > about 1.095 g / ml, which is referred to as "B5". In some embodiments, the cell population enriched with tubular cells is relatively depleted of all of the following: "B1", "B5", oxygen-tunable EPO-expressing cells, glomerular cells, and vascular cells.

[0044] As used herein, the term “hypoxic” culture conditions refers to culture conditions in which the level of oxygen available to cells in a culture system is reduced compared to standard culture conditions in which cells are cultured at atmospheric oxygen levels (about 21%). Non-hypoxic conditions refer to normal or standard culture conditions in this specification.

[0045] The term "oxygen-tunable" as used herein refers to the ability of a cell to regulate (up or down) gene expression based on the amount of oxygen available to the cell. "Hypoxia-inducible" refers to the upregulation of gene expression in response to a decrease in oxygen negative pressure (regardless of pre-induced or initial oxygen partial pressure).

[0046] The term “biomaterial” as used herein refers to a natural or synthetic biocompatible material suitable for injection into living tissue. Natural biomaterial is a material produced by or originating from the biological system. Synthetic biomaterial is a material not produced by or originating from the biological system. The biomaterial disclosed herein may be a combination of natural and synthetic biocompatible materials. As used herein, biomaterial includes, for example, a polymeric matrix and a scaffold. A person skilled in the art will recognize that the biomaterial(s) may be formed in various forms, for example, as a porous foam, gel, liquid, bead, or solid, and may comprise one or more natural or synthetic biocompatible materials. In one embodiment, the biomaterial is a solution in liquid form that may be a hydrogel.

[0047] The term "modified release" or the equivalent terms "controlled release," "delayed release," or "sustained release" refers to a formulation that releases an activator, such as bioactive cells, over a period of time or at more than one time point after administration to an individual. Depending on the formulation, the modified release of the activator, which may occur over a preferred time range—e.g., minutes, hours, days, weeks, or longer—is contrasted with standard formulations where substantially the entire dose unit is available immediately after administration. For tissue manipulation and regenerative medicine applications, a preferred modified-release formulation provides the release of the activator at multiple time points after local administration (e.g., administering the activator directly to a solid organ). For example, a modified-release formulation of bioactive cells will provide the initial release of the cells immediately at the time of administration, followed by a secondary release. The time delay for the secondary release of the activator may be minutes, hours, or days after the initial administration. Generally, the time duration for the release delay corresponds to the duration of time required for the biomaterial carrier of the activator to lose its structural integrity. The delayed release of the activator begins, and consequently, integrity starts to decompose and is completed at the time when integrity completely collapses. A person skilled in the art will recognize the appropriate mechanism of release.

[0048] The term “anemia” as used herein refers to a deficiency in the number of red blood cells and / or hemoglobin levels resulting from the inadequate production of functional EPO proteins by the subject’s EPO-producing cells and / or inadequate release of EPO proteins into the systemic circulation and / or incapable response of bone marrow erythroblasts to EPO proteins. A subject with anemia is unable to maintain red blood cell homeostasis. Generally, anemia may occur due to a decline or loss of renal function (e.g., chronic renal failure), anemia associated with relative EPO deficiency, anemia associated with congestive heart failure, anemia associated with myelosuppressive therapy such as chemotherapy or antiviral therapy (e.g., AZT), anemia associated with non-myeloid cancer, anemia associated with viral infections such as HIV, and anemia associated with chronic diseases such as autoimmune diseases (e.g., rheumatoid arthritis), liver disease, and multiple organ system failure.

[0049] The term "EPO-deficiency" refers to any condition or disorder treatable with an erythropoetin receptor agonist (e.g., recombinant EPO or EPO analogs), including anemia.

[0050] The term “organ-related disease” as used herein refers to a disorder associated with any stage or degree of acute or chronic organ failure that causes a loss of the organ’s ability to perform organ functions.

[0051] The term “renal disease” as used herein refers to any stage or degree of acute or chronic renal failure that causes a loss of the kidney’s ability to filter blood and remove excess fluid, electrolytes, and waste from the blood. Renal disease also includes endocrine dysfunctions such as anemia (erythropoietin deficiency) and mineral imbalances (vitamin D deficiency). Renal disease may originate in the kidneys or may be secondary to various conditions including (but not limited to) heart failure, hypertension, diabetes, autoimmune diseases, or liver disease. Renal disease may be a state of chronic renal failure that develops after acute injury to the kidneys. For example, kidney injury caused by ischemia and / or toxic exposure can cause acute renal failure; and incomplete recovery after acute kidney injury can lead to the development of chronic renal failure.

[0052] The term “treatment” refers to any means of treating, preventing, or preventing renal disease, anemia, EPO deficiency, tubular transport deficiency, or glomerular filtration deficiency, the purpose of which is to reverse, prevent, or delay (alleviate) the target disease. Persons requiring such treatment include those who already have renal disease, anemia, EPO deficiency, tubular transport deficiency, or glomerular filtration deficiency, and those who are predisposed to having renal disease, anemia, EPO deficiency, tubular transport deficiency, or glomerular filtration deficiency, or those who need to prevent renal disease, anemia, EPO deficiency, tubular transport deficiency, or glomerular filtration deficiency. As used herein, the term “treatment” includes the stabilization and / or improvement of renal function.

[0053] The term “in vivo contact” as used herein refers to direct in vivo contact between a product secreted by a concentrated population of cells and the proper organ. For example, a product secreted by a concentrated population of kidney cells (or a mixture or a structure containing kidney cells / kidney cell fractions) may have in vivo contact with the proper kidney. Direct in vivo contact may actually be paracrine, endocrine, or lateral. The secreted product may be a heterogeneous population of different products described herein.

[0054] As used herein, the terms “ribonucleic acid” or “RNA” refer to a chain of nucleotide units, each unit consisting of a nitrogenous base, ribose sugar, and phosphate. RNA may be in a single or double-stranded form. RNA may be part of, contained in, or bound to a vesicle. The vesicle may be an exosome. RNA includes, but is not limited to, mRNA, rRNA, small RNA, snRNA, snoRNA, microRNA (miRNA), small interfering RNA (siRNA), and non-coding RNA. Preferably, RNA is human RNA.

[0055] The term “structure” refers to one or more cell populations deposited on or within a scaffold or matrix prepared from one or more synthetic or naturally occurring biocompatible materials. One or more cell populations may be coated with a biomaterial prepared from one or more synthetic or naturally occurring biocompatible materials, polymers, proteins, or peptides, and may be deposited on, embedded within, attached to, smeared within, or captured within the biomaterial. One or more cell populations may be combined with the biomaterial or scaffold or matrix in vitro or in vivo. Generally, one or more biocompatible materials used to form the scaffold / biomaterial are selected to direct, promote, or allow the formation of a multicellular three-dimensional structure of at least one cell population deposited thereon. One or more biomaterials used to create the structure may also be selected to direct, promote, or allow the dispersion and / or integration of the structure or the cellular components of the structure with endogenous host tissues, or to direct, promote, or allow the survival, grafting, tolerance, or performance of function of the structure or the cellular components of the structure.

[0056] The terms “marker” or “biomarker” generally refer to DNA, RNA, protein, carbohydrate, or glycolipid-based molecular markers, the expression or presence thereof in a cultured cell population can be detected by standard methods (or methods disclosed herein) and correspond to one or more cells of a specific cell type in a cultured cell population. These biomarkers are [Table] X and YThe marker includes, but is not limited to, the genes presented in [the example]. The marker may be a polypeptide expressed by a cell or an identifiable physical location on a chromosome, e.g., a gene, a restriction endonuclease recognition site, or a nucleic acid encoding a polypeptide expressed by the cell (e.g., mRNA). The marker may be an expression region of a gene referred to as a "gene expression marker" or a fragment of DNA having an unknown coding function. The biomarker may be a cell-derived product, e.g., a secreted product.

[0057] The terms “biomarker signature,” “signature,” “biomarker expression signature,” or “expression signature” are used interchangeably herein and represent a single biomarker or a combination thereof, wherein the expression of the biomarker is an indicator of cell type(s), e.g., cell population, e.g., epithelial cells, tubular cells, etc., including bioactive renal cells. The biomarker signature may serve as an indicator of the suitability of the cell population for use in the methods and preparations provided herein. In some embodiments, the biomarker signature is a “gene signature.” The term “gene signature” is used interchangeably with “gene expression signature” and represents a single polynucleotide or a combination thereof, wherein the expression of the polynucleotide is an indicator of cell type, e.g., epithelial cells, tubular cells, etc. In some embodiments, the biomarker signature is a “protein signature.” The term "protein signature" is used interchangeably with "protein expression signature" and refers to a single polypeptide or a combination thereof, wherein the expression of the polypeptide is an indicator of the cell type, such as epithelial cells or tubular cells.

[0058] The terms "level of expression" and "level of expression" are used interchangeably and generally refer to the amount of polynucleotide or amino acid products in a biological sample or the percentage of cells expressing polynucleotide or amino acid products or proteins. "Expressing" or "expression" and its grammatical variations indicate the presence of detectable amounts of polynucleotide or amino acid products or proteins in a biological sample. For example, it may be referred to as expressing a detectable (background or control value) protein. Similarly, if some of the cells in a sample express a protein, the sample may be referred to as expressing a protein. Alternatively, the sample may be referred to as having a level of expression related to the percentage of cells expressing a protein; for example, if 60% of the cells in the sample express a protein, the level of expression is 60%.

[0059] The terms “differently expressed gene,” “differential gene expression,” and their synonyms, used interchangeably, refer to a gene that is activated to a higher or lower level of expression in a first cell or cell population compared to expression in a second cell or cell population. The term also includes a gene that is activated to a higher or lower level of expression at different stages over time during passages of the first or second cell in culture. Additionally, it is understood that a differentially expressed gene may be activated or repressed at the nucleic acid level or the protein level, or may be alternatively spliced ​​to produce different polypeptide products. Such differences may be demonstrated, for example, by changes in mRNA levels, surface expression, secretion, or other divisions of the polypeptide. Differential gene expression may include a comparison of expression between two or more genes or their gene products, a comparison of expression ratios between two or more genes or their gene products, or even a comparison of two differently processed products of the same gene that differ between the first cell and the second cell. Differential expression includes both quantitative and qualitative differences in the temporal or cellular expression patterns of a gene or its expression products, for example, between a first cell and a second cell. For the purposes of this disclosure, if there is a difference between the expression of a given gene in a first cell and a second cell, "differential gene expression" is considered to exist. Differential expression of a marker may be present in cells from a patient prior to administration of the (first cell) cell population, mixture, or construct, compared to expression in cells from the patient after administration of the (second cell).

[0060] The terms "inhibit," "downregulate," "downexpress," and "reduce" are used interchangeably and mean that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits is reduced compared to one or more controls, e.g., one or more positive and / or negative controls. Downexpression may be present in cells from a patient prior to administration of the cell population, mixture, or construct, compared to cells from the patient after administration.

[0061] The terms "up-regulate" or "over-express" mean that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits is increased compared to one or more controls, e.g., one or more positive and / or negative controls. Over-expression may be present in cells from a patient after administration of a cell population, mixture, or construct compared to cells from the patient before administration.

[0062] The term “subject” means any single human subject, including a patient who is eligible for treatment and who has experienced or has experienced one or more signs, symptoms, or other indicators of organ-related diseases such as renal disease, anemia, or EPO deficiency. Such subjects include, but are not limited to, subjects who have been newly diagnosed or previously diagnosed with renal disease, anemia, or EPO deficiency, regardless of cause, and who have experienced a relapse or exacerbation or are at risk of developing these diseases. Subjects may or may not have previously received treatment for renal disease, anemia, or EPO deficiency.

[0063] The term "patient" refers to any single animal seeking treatment, more preferably a mammal (including, for example, non-human animals such as dogs, cats, horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates). Most preferably, the patient is a human.

[0064] The terms "sample," "patient sample," or "biological sample" generally refer to any biological sample obtained from a subject or patient, body fluid, body tissue, cell line, tissue culture, or other source. This term includes tissue biopsies, such as, for example, a kidney biopsy. This term includes cultured cells, such as, for example, cultured mammalian kidney cells. Methods for obtaining tissue biopsies and cultured cells from mammals are well known in the field. If the term "sample" is used alone, it still means "biological sample" or "patient sample"; that is, these terms are used interchangeably.

[0065] The term "test sample" refers to a sample from a subject processed by the method disclosed in the present invention. The test sample may originate from various sources of mammalian subjects, including but not limited to blood, semen, serum, urine, bone marrow, mucous membranes, tissues, etc.

[0066] The terms “control” or “control sample” refer to a negative or positive control expected to yield a negative or positive result that helps demonstrate the association of the results of the test sample. Controls suitable for the present invention include, but are not limited to, samples known to exhibit characteristic indicators of normal erythrocyte homeostasis, samples known to exhibit characteristic indicators of anemia, samples obtained from subjects known not to have anemia, and samples obtained from subjects known to have anemia. Additional controls suitable for use in the methods provided herein include, but are not limited to, samples derived from subjects treated with pharmacological substances known to regulate erythrocyte production (e.g., recombinant EPO or EPO analogs). Additionally, the control may be a sample obtained from a subject prior to treatment with the method disclosed herein. Further suitable controls may be a test sample obtained from a subject known to have renal disease of any type or stage, and a sample from a subject known not to have renal disease of any type or stage. The control may be a normal, healthy matched control. A person skilled in the art will recognize other comparisons suitable for use in this specification.

[0067] "Regenerative prognosis," "regenerative prognosis," or "prognosis for regeneration" generally refers to a prediction or forecast of a promising regenerative process or outcome from the administration or transplantation of the cell population, mixture, or structure described herein. In the case of a regenerative prognosis, the prediction or forecast may be known by one or more of the following: improvement of a functional organ (e.g., kidney) after transplantation or administration; development of a functional kidney after transplantation or administration; development of improved kidney function or capacity after transplantation or administration; and expression of specific markers by the intrinsic kidney after transplantation or administration.

[0068] "Regenerated organ" refers to an organ after the transplantation or administration of a cell population, mixture, or structure as described herein. The regenerated organ is characterized by various indicators, including but not limited to the development of function or capacity in the organ, improvement of function or capacity in the organ, and expression of specific markers in the organ. A person skilled in the art will recognize that other indicators are suitable for characterizing the regenerated organ.

[0069] "Regenerated kidney" refers to an intrinsic kidney following the transplantation or administration of a cell population, mixture, or structure as described herein. The regenerated kidney is characterized by various indicators, including but not limited to the development of function or capacity in the intrinsic kidney, the improvement of function or capacity in the intrinsic kidney, and the expression of specific markers in the intrinsic kidney. A person skilled in the art will recognize that other indicators may also be suitable for characterizing the regenerated kidney.

[0070] The term "cell aggregate" or "spheroid" refers to an aggregate or assembly of cells cultured to allow for 3D growth, as opposed to growth as a monolayer. Note that the term "spheroid" implies that the aggregate is not a geometric sphere. The aggregate may be highly organized with a well-defined morphology or may be an unorganized mass; the aggregate may contain a single cell type or more than one cell type. The cells may be primary isolates, permanent cell lines, or a combination of both. Organoids and organotypic cultures are included in this definition.

[0071] The term “ambient temperature” refers to the temperature at which the formulation of the present disclosure is administered to a subject. Generally, the ambient temperature is the temperature of a temperature-controlled environment. The ambient temperature ranges from about 18°C ​​to about 30°C. In one embodiment, the ambient temperature is about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, or about 30°C.

[0072] As used herein, the term “label” refers to a compound or composition that is directly or indirectly conjugated or fused with a reagent, such as a nucleic acid probe or antibody, and facilitates the detection of the conjugated or fused reagent. The label may be detectable in itself (e.g., radioisotope label or fluorescent label) or, in the case of an enzyme label, catalyze the chemical modification of a detectable substrate compound or composition. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) the probe or antibody with a detectable substance, and indirect labeling of a probe or antibody by reactivity to another directly labeled reagent. Examples of indirect labeling include the detection of a primary antibody using a fluorescently labeled secondary antibody and the terminal labeling of a DNA probe with biotin so that it can be detected by fluorescently labeled streptavidin.

[0073] The term "detection" includes any means of detection, including direct and indirect detection.

[0074] The “kit” is any manufactured product (e.g., package or container) comprising at least one reagent, e.g., a drug for the treatment of kidney disease, or a probe for specifically detecting a biomarker gene or protein as disclosed herein. The manufactured product is preferably promoted, distributed, or sold as a unit for carrying out the method disclosed herein.

[0075] 2. Cell population

[0076] The formulations of the present disclosure may contain an isolated, heterogeneous population of kidney cells and a mixture thereof, in which a specific bioactive component or cell type is concentrated and / or a specific inactive or undesirable component or cell type is depleted, for use in the treatment of kidney disease, i.e., to provide stabilization and / or improvement and / or regeneration of kidney function, and Presnell et al . US 2011-0117162 and Ilagan et al . Previously described in PCT / US2011 / 036347, the entire contents of which are incorporated herein by reference. The formulation may contain isolated renal cell fractions that lack cellular components compared to healthy subjects but possess therapeutic properties, namely, providing stabilization and / or improvement and / or regeneration of renal function. The cell populations, cell fractions, and / or mixtures of cells described herein may be derived from healthy subjects, subjects with renal disease, or subjects described herein.

[0077] The present disclosure provides a formulation suitable for use with various bioactive cell populations, comprising, but not limited to, isolated cell population(s), cell fraction(s), mixture(s), concentrated cell population(s), cell aggregate(s), and any combination thereof. In a specific example, the bioactive cell population is a bioactive kidney cell.

[0078] Bioactive cell population

[0079] The present disclosure considers therapeutic formulations suitable for bioactive cell populations to be administered to target organs or tissues within a subject requiring bioactive cell populations. A bioactive cell population generally refers to a cell population that potentially possesses therapeutic properties when administered to a subject. For example, when administered to a subject requiring such a population, a bioactive renal cell population may provide stabilization and / or improvement and / or regeneration of renal function in the subject. Therapeutic properties may include regenerative effects.

[0080] The biologically active cell population comprises, but is not limited to, stem cells (e.g., pluripotent, multipotent, micropotent, or unipotent) such as embryonic stem cells, amniotic stem cells, adult stem cells (e.g., hematopoietic, breast, intestinal, mesenchymal, placental, lung, bone marrow, blood, umbilical cord, endothelium, pulp, fat, nerve, olfactory, neural tube, testis); genetically modified cells; and cell populations or tissue explants derived from any source of the body. The formulations of the present disclosure also include, Basu filed on June 9, 2011 et al . together with a renal adipose-derived cell population as described in PCT / US11 / 39859; and Ludlow submitted on May 3, 2011 et al . US 2010-0131075 and Ludlow et al . It may be used with adipose-derived or peripheral blood-derived smooth muscle as described in PCT / US11 / 35058; or with bladder-derived urothelium or smooth muscle as described in Atala US 6,576,019, each of which is incorporated herein by reference in its entirety. The bioactive cell population may be isolated from nature, concentrated, purified, homogeneous, or heterogeneous. A person skilled in the art will recognize other bioactive cell populations suitable for use in the formulations of the present disclosure.

[0081] In one embodiment, the source of the cells is the same as the intended target organ or tissue. For example, kidney cells may be sourced from the kidney to be used in a preparation to be administered to the kidney. In another embodiment, the source of the cells is not the same as the intended target organ or tissue. For example, erythropoietin-expressing cells may be sourced from kidney fat to be used in a preparation to be administered to the kidney.

[0082] In one aspect, the present disclosure provides that a formulation containing a specific subfraction of a heterogeneous population of kidney cells, in which the bioactive component is concentrated and the inactive or undesirable component is depleted, provides therapeutic and regenerative results superior to those of the initial cell population. For example, the bioactive kidney cells described herein, e.g., B2, B4, and B3, in which the inactive or undesirable component, e.g., B1 and B5, are depleted, may be part of a formulation to be used for the stabilization and / or improvement and / or regeneration of kidney function, either alone or in combination.

[0083] In another aspect, the formulation contains B4, a specific subfraction of one or more cell types, e.g., vascular, endocrine, or endothelium, in which B4' is depleted or deficient, which, alone or when mixed with other bioactive subfractions, e.g., B2 and / or B3, possesses therapeutic properties, e.g., stabilization and / or improvement and / or regeneration of renal function. In a preferred embodiment, the bioactive cell population is B2. In a specific embodiment, the B2 cell population is mixed with B4 or B4'. In another embodiment, the B2 cell population is mixed with B3. In another embodiment, the B2 cell population is mixed with both B3 and B4, or with specific cellular components of B3 and / or B4.

[0084] The B2 cell population is characterized by the expression of tubular cell markers selected from a group consisting of one or more of the following: megalin, curbilin, hyaluronic acid synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, member of the RAS oncogene family (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9 (sodium / hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3 (Aldh1a3), and calpain-8 (Capn8), and collecting duct markers. Aquaporin-4 (Aqp4). B2 is larger and more granulated than B3 and / or B4, having a buoyancy density of about 1.045 g / ml to about 1.063 g / ml (rodents), about 1.045 g / ml to 1.052 g / ml (humans), and about 1.045 g / ml to about 1.058 g / ml (animals).

[0085] The B3 cell population is characterized by the expression of vascular, glomerular, and proximal tubular markers along with some EPO-producing cells, and has intermediate size and particle size compared to B2 and B4, with buoyancy densities of about 1.063 g / ml to about 1.073 g / ml (rodents), about 1.052 g / ml to about 1.063 g / ml (humans), and about 1.058 g / ml to about 1.063 g / ml (individuals). B3 is characterized by the expression of markers selected from the group consisting of one or more of the following: aquaporin 7 (Aqp7), FXYD domain-containing ion transport regulator 2 (Fxyd2), solute carrier family 17 (sodium phosphate), member 3 (Slc17a3), solute carrier family 3, member 1 (Slc3a1), claudin 2 (Cldn2), napsin A aspartic peptidase (Napsa), solute carrier family 2 (facilitated glucose transporter), member 2 (Slc2a2), alanyl (membrane) aminopeptidase (Anpep), transmembrane protein 27 (Tmem27), acyl-CoA synthetase medium-chain family member 2 (Acsm2), glutathione peroxidase 3 (Gpx3), fructose-1,6-biphosphatase 1 (Fbp1), and Alanine-glyoxylate aminotransferase 2 (Agxt2). B3 is also characterized by the vascular expression marker platelet endothelial cell adsorption molecule (Pecam) and the glomerular expression marker dodn (Podn).

[0086] The B4 cell population is characterized by the expression of an oxygen-tunable EPO-rich population compared to undivided (UNFX), B2, and B3, and a set of vascular markers containing one or more of the following: PECAM, VEGF, KDR, HIF1a, CD31, CD146; a set of glomerular markers containing one or more of the following: Podn (Podn) and Nephrin (Neph); and undivided (UNFX). B4 is also characterized by the expression of one or more of the following markers: chemokine (CXC motif) receptor 4 (Cxcr4), endothelin receptor type B (Ednrb), collagen, type V, alpha 2 (Col5a2), cadherin 5 (Cdh5), plasminogen activator, tissue (Plat), angiopoietin 2 (Angpt2), kinase insertion domain protein receptor (Kdr), secreted protein, acidic cysteine-rich (osteonectin) (Sparc), serglycine (Srgn), TIMP metallopeptidase inhibitor 3 (Timp3), Wilms tumor 1 (Wt1), wingless-type MMTV integration site family, member 4 (Wnt4), regulator of G-protein signaling 4 (Rgs4), platelet-endothelial cell adsorption molecule (Pecam), and erythropoietin (Epo). B4 is also characterized by smaller, less granulated cells compared to B2 or B3, where the buoyancy density is about 1.073 g / ml to about 1.091 g / ml (rodents) and about 1.063 g / ml to about 1.091 g / mL (humans and dogs).

[0087] A B4' cell population is defined as having a buoyancy density of 1.063 g / mL to 1.091 g / mL and expressing one or more of the following markers: PECAM, vEGF, KDR, HIF1a, dodn, nephrine, EPO, CK7, CK8 / 18 / 19. In one embodiment, the B4' cell population is characterized by the expression of a set of vascular markers containing one or more of the following: PECAM, vEGF, KDR, HIF1a, CD31, CD146. In another embodiment, the B4' cell population is characterized by the expression of the endocrine marker EPO. In one embodiment, the B4' cell population is characterized by the expression of a set of glomerular markers containing one or more of the following: dodn, and nephrine. In a specific embodiment, the B4' cell population is characterized by the expression of a set of vascular markers containing one or more of PECAM, vEGF, KDR, and HIF1a, and by the expression of the endocrine marker EPO. In another embodiment, B4' is also characterized as smaller, less granulated cells compared to B2 or B3, where the buoyancy density is about 1.073 g / ml to about 1.091 g / ml (rodents), about 1.063 g / ml to about 1.091 g / mL (humans and dogs).

[0088] In one aspect, the present disclosure provides a formulation containing an isolated, concentrated B4' cell population of human kidney cells comprising at least one of erythropoietin (EPO)-producing cells, vascular cells, and glomerular cells, having a density of 1.063 g / mL to 1.091 g / mL. In one embodiment, the B4' cell population is characterized by the expression of a vascular marker. In a specific embodiment, the B4' cell population is not characterized by the expression of a glomerular marker. In some embodiments, the B4' cell population may exhibit oxygen-tunable erythropoietin (EPO) expression.

[0089] In one embodiment, the formulation contains a B4' cell population but does not contain a B2 cell population including tubular cells having a density of 1.045 g / mL to 1.052 g / mL. In another embodiment, the B4' cell population formulation does not contain a B1 cell population including large granule cells of the collecting duct and tubular system having a density of < 1.045 g / mL. In another embodiment, the B4' cell population formulation does not contain a B5 cell population including fragments and small cells of low particle size and viability having a density of > 1.091 g / mL.

[0090] In one embodiment, the B4' cell population-containing formulation does not include a B2 cell population comprising tubular cells having a density of 1.045 g / mL to 1.052 g / mL; a B1 cell population comprising large granular cells of the collecting duct and tubular system having a density of < 1.045 g / mL; and a B5 cell population comprising fragments and small cells of low particle size and viability having a density of > 1.091 g / mL. In some embodiments, the B4' cell population may be derived from a subject having kidney disease.

[0091] In one aspect, the present disclosure provides a formulation containing a mixture of human kidney cells comprising a first cell population, B2, comprising an isolated, concentrated population of tubular cells having a density of 1.045 g / mL to 1.052 g / mL, and a second cell population, B4', comprising erythropoietin (EPO)-producing cells and vascular cells having a density of about 1.063 g / mL to 1.091 g / mL, but depleted of glomerular cells, wherein the mixture does not include a B1 cell population comprising large granule cells of the collecting duct and tubular system having a density of < 1.045 g / mL, or a B5 cell population comprising fragments and small molecules of low particle size and viability having a density of > 1.091 g / mL. In a specific embodiment, the B4' cell population is characterized by the expression of a vascular marker. In one embodiment, the B4' cell population is not characterized by the expression of a glomerular marker. In a specific embodiment, B2 further comprises collecting duct epithelial cells. In one embodiment, the formulation contains a mixture of cells capable of receptor-mediated albumin uptake. In another embodiment, the mixture of cells may express oxygen-tunable erythropoietin (EPO). In one embodiment, the mixture contains HAS-2-expressing cells capable of generating and / or stimulating the production of high molecular weight hyaluronic acid (HA) species both in vitro and in vivo. In all embodiments, the first and second cell populations may be derived from kidney tissue or cultured kidney cells (Basu et al . Lipids in Health and Disease, 2011, 10:171).

[0092] In one embodiment, the formulation contains a mixture capable of providing regenerative stimulation upon in vivo delivery. In another embodiment, the mixture may reduce, stabilize, or improve the decline in glomerular filtration, tubular reabsorption, urine production, and / or endocrine function upon in vivo delivery. In one embodiment, the B4' cell population is derived from a subject with kidney disease.

[0093] In one aspect, the present disclosure provides a formulation containing an isolated, concentrated B4' population of human kidney cells comprising at least one of erythropoietin (EPO)-producing cells, vascular cells, and glomerular cells having a density of 1.063 g / mL to 1.091 g / mL. In one embodiment, the B4' cell population is characterized by the expression of a vascular marker. In a specific embodiment, the B4' cell population is not characterized by the expression of a glomerular marker. The glomerular marker that is not expressed may be grapesine. In some embodiments, the B4' cell population may have oxygen-tunable erythropoietin (EPO) expression.

[0094] In one embodiment, the B4' cell population-containing formulation does not contain a B2 cell population including tubular cells having a density of 1.045 g / mL to 1.052 g / mL. In another embodiment, the B4' cell population formulation does not contain a B1 cell population including large glomerular cells of the collecting duct and tubular system having a density of < 1.045 g / mL. In another embodiment, the B1 cell population formulation does not contain a B5 cell population including fragments and small cells of low particle size and viability having a density of > 1.091 g / mL.

[0095] In one embodiment, the B4' cell population-containing formulation does not include a B2 cell population comprising tubular cells having a density of 1.045 g / mL to 1.052 g / mL; a B1 cell population comprising large granular cells of the collecting duct and tubular system having a density of < 1.045 g / mL; and a B5 cell population comprising fragments and small cells of low particle size and viability having a density of > 1.091 g / mL. In some embodiments, the B4' cell population may be derived from a subject having kidney disease.

[0096] In one aspect, the present disclosure provides a formulation containing a mixture of human kidney cells comprising a first cell population, B2, comprising an isolated, concentrated population of tubular cells having a density of 1.045 g / mL to 1.052 g / mL, and a second cell population, B4', comprising erythropoietin (EPO)-producing cells and vascular cells having a density of about 1.063 g / mL to 1.091 g / mL but depleted of glomerular cells, wherein the mixture does not include a B1 cell population comprising large granule cells of the collecting duct and tubular system having a density of < 1.045 g / mL, or a B5 cell population comprising fragments and small cells having low particle size and viability having a density of > 1.091 g / mL. In a specific embodiment, the B4' cell population is characterized by the expression of a vascular marker. In one embodiment, the B4' cell population is not characterized by the expression of a glomerular marker. In a specific embodiment, B2 further comprises collecting duct epithelial cells. In one embodiment, the cell mixture may undergo receptor-mediated albumin uptake. In another embodiment, the cell mixture may undergo oxygen-tunable erythropoietin (EPO) expression. In one embodiment, the mixture contains HAS-2-expressing cells capable of generating and / or stimulating the production of high molecular weight hyaluronic acid (HA) species both in vitro and in vivo. In all embodiments, the first and second cell populations may be derived from kidney tissue or cultured kidney cells.

[0097] In another aspect, the present disclosure provides a formulation containing a heterogeneous renal cell population comprising a combination of cell fractions or concentrated cell populations (e.g., B1, B2, B3, B4 (or B4'), and B5). In one embodiment, the combination has a buoyancy density of about 1.045 g / ml to about 1.091 g / ml. In one other embodiment, the combination has a buoyancy density of 1.045 g / ml to less than about 1.099 g / ml or about 1.100 g / ml. In another embodiment, the combination has a buoyancy density as determined by separation on a density gradient, e.g., by centrifugation. In another embodiment, the combination of cell fractions contains B2, B3, and B4 (or B4') depleted of B1 and / or B5. In some embodiments, the combination of cell fractions contains B2, B3, B4 (or B4') and B5, but B1 is depleted. Once B1 and / or B5 is depleted, the combination may be subsequently cultured in vitro prior to the preparation of a formulation containing a combination of B2, B3, and B4 (or B4') cell fractions.

[0098] The inventors of the present disclosure have surprisingly discovered that in vitro culture of a B1-depleted combination of B2, B3, B4, and B5 causes depletion of B5. In one embodiment, B5 is depleted after at least 1, 2, 3, 4, or 5 passages. In one other embodiment, a combination of B2, B3, B4, and B5 cell fractions passaged under the conditions described herein provides a passaged cell population having B5 in a percentage of less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.5%.

[0099] In another embodiment, B4' is part of a combination of cell fractions. In one other embodiment, in vitro culture depletion of B5 occurs under hypoxic conditions.

[0100] In one embodiment, the formulation contains a mixture capable of providing regenerative stimulation upon in vivo delivery. In another embodiment, the mixture may reduce, stabilize, or improve the decline in glomerular filtration, tubular reabsorption, urine production, and / or endocrine function upon in vivo delivery. In one embodiment, the B4' cell population is derived from a subject with renal disease.

[0101] In a preferred embodiment, the formulation contains a mixture comprising B2 in combination with B3 and / or B4. In another preferred embodiment, the mixture comprises B2 in combination with B3 and / or B4'. In another preferred embodiment, the mixture is composed of or essentially composed of (i) B2 in combination with B3 and / or B4; or (ii) B2 in combination with B3 and / or B4'.

[0102] A mixture containing a B4' cell population may also contain a B2 and / or B3 cell population obtained from a non-healthy subject. The non-healthy subject may be the same subject from which the B4' fraction was obtained. In contrast to the B4' cell population, the B2 and B3 cell populations obtained from the non-healthy subject are typically not deficient in one or more specific cell types compared to the initial kidney cell population derived from a healthy individual.

[0103] Presnell et alAs described in WO / 2010 / 056328, it was found that B2 and B4 cell products can express larger molecular weight hyaluronic acid (HA) species both in vitro and in vivo through the action of hyaluronic acid synthase-2 (HAS-2)—a marker that is more specifically concentrated in B2 cell populations. In a 5 / 6 Nx model, treatment with B2 showed a reduction in fibrosis, and simultaneously, strong HAS-2 expression in vivo and the production of high molecular weight HA within the treated tissues were expected. In particular, the untreated 5 / 6 Nx model resulted in fibrosis with limited detection of HAS-2, and almost no high molecular weight HA was produced. Without being limited to theory, it is hypothesized that anti-inflammatory high molecular weight HA species, primarily produced by B2 (and to some extent B4), act synergistically with cell products in the reduction of renal fibrosis and regeneration. Accordingly, the present disclosure comprises a preparation containing bioactive kidney cells as described herein, together with a biomaterial including hyaluronic acid. The provision of a biomaterial component for regenerative stimulation through direct generation by transplanted cells or stimulation of generation is also considered in the present disclosure.

[0104] In one aspect, the present disclosure provides a formulation containing an isolated heterogeneous population of EPO-producing kidney for use in treating kidney disease, anemia, and / or EPO deficiency in subjects requiring such treatment. In one embodiment, the cell population is derived from a kidney biopsy. In another embodiment, the cell population is derived from whole kidney tissue. In another embodiment, the cell population is derived from an in vitro culture of mammalian kidney cells established from a kidney biopsy or whole kidney tissue. In all embodiments, these populations are undivided cell populations, which are also referred to herein as non-enriched cell populations.

[0105] In another aspect, the present disclosure provides a formulation containing an isolated population of erythropoietin (EPO)-producing renal cells that is further enriched such that the proportion of EPO-producing cells in the enriched subpopulation is greater than the proportion of EPO-producing cells in the initial or first cell population. In one embodiment, the enriched EPO-producing cell fraction contains a larger proportion of interstitial fibroblasts and a smaller proportion of tubular cells compared to the interstitial fibroblasts and tubular cells contained in the non-enriched initial population. In a specific embodiment, the enriched EPO-producing cell fraction contains a larger proportion of glomerular cells and vascular cells and a smaller proportion of collecting duct cells compared to the glomerular cells, vascular cells, and collecting duct cells contained in the non-enriched initial population. In these embodiments, these populations are also referred to herein as the “B4” cell population.

[0106] In another aspect, the present disclosure provides a formulation comprising an EPO-producing renal cell population mixed with one or more additional renal cell populations. In one embodiment, the EPO-producing cell population is a first cell population in which EPO-producing cells are concentrated, e.g., It is B4. In another embodiment, the EPO-producing cell population is a first cell population, e.g., B2, in which EPO-producing cells are not enriched. In another embodiment, the first cell population is mixed with a second renal cell population. In some embodiments, the second cell population is enriched with tubular cells, which can be demonstrated by the presence of a tubular cell phenotype. In another embodiment, the tubular cell phenotype can be specified by the presence of a single tubular cell marker. In another embodiment, the tubular cell phenotype can be specified by the presence of one or more tubular cell markers. Tubular cell markers include, but are not limited to, megalin, curbilin, hyaluronic acid synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, member RAS oncogene family (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9 (sodium / hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3 (Aldh1a3), and calpain-8 (Capn8). In another embodiment, the first cell population is mixed with at least one of several renal cell types, including but not limited to interstitial-derived cells, tubular cells, collecting duct-derived cells, glomerular-derived cells, and / or cells derived from blood or vascular structures.

[0107] The formulation of the present disclosure may comprise an EPO-producing kidney cell population containing B4 or B4' in the form of a mixture with B2 and / or B3, or in the form of a concentrated cell population, e.g., B2+B3+B4 / B4'.

[0108] In one aspect, the formulation contains an EPO-producing kidney cell population characterized by EPO expression and bioresponsiveness to oxygen, such that a decrease in the partial pressure of oxygen in the culture system causes the induction of EPO expression. In one embodiment, the EPO-producing cell population is enriched with EPO-producing cells. In one embodiment, EPO expression is induced when the cell population is cultured under conditions where the level of oxygen available in the culture system is reduced compared to a cell population cultured at normal atmospheric levels of available oxygen (~21%). In one embodiment, EPO-producing cells cultured under lower oxygen conditions express greater levels of EPO compared to EPO-producing cells cultured under normal oxygen conditions. Generally, culturing cells at reduced levels of available oxygen (also referred to as hypoxic culture conditions) means that the level of oxygen is reduced compared to culturing cells at normal atmospheric levels of available oxygen (also referred to as normal or standard oxygen culture conditions). In one embodiment, hypoxic cell culture conditions include culturing cells at about 1% oxygen, about 2% oxygen, about 3% oxygen, about 4% oxygen, or about 5% oxygen. In another embodiment, normal or standard oxygen culture conditions include culturing cells at about 10% oxygen, about 12% oxygen, about 13% oxygen, about 14% oxygen, about 15% oxygen, about 16% oxygen, about 17% oxygen, about 18% oxygen, about 19% oxygen, about 20% oxygen, or about 21% oxygen.

[0109] In one other embodiment, the induction or increased expression of EPO can be obtained and observed by culturing cells at less than about 5% available oxygen and comparing EPO levels with those of cells cultured at atmospheric oxygen (about 21%). In another embodiment, the induction of EPO is obtained by culturing cells capable of expressing EPO by a method comprising a first culture step in which the cells are cultured at atmospheric oxygen (about 21%) for some time, and a second culture step in which the same cells are cultured at a reduced level of available oxygen and less than about 5% available oxygen. In another embodiment, EPO expression in response to hypoxic conditions is regulated by HIF1α. A person skilled in the art will recognize that other oxygen-regulated culture conditions known in the art may be used for the cells described herein.

[0110] In one aspect, the formulation contains a concentrated population of EPO-producing mammalian cells characterized by bio-responsiveness to perfusion conditions (e.g., EPO expression). In one embodiment, perfusion conditions include transient, intermittent, or continuous fluid flow (perfusion). In one embodiment, EPO expression is mechanically induced when the medium in which the cells are cultured is circulated intermittently or continuously or stirred, in such a manner where power is delivered to the cells through the flow. In one embodiment, cells undergoing transient, intermittent, or continuous fluid flow are cultured in such a manner where they exist as three-dimensional structures or are present within or on a material that provides a framework and / or space for forming these three-dimensional structures. In one embodiment, the cells are cultured on porous beads and receive intermittent or continuous fluid flow through a shaking platform, an orbiting platform, or a spinner flask. In another embodiment, cells are cultured in a three-dimensional scaffold, the scaffold is fixed, and a device is placed such that a fluid flows directionally through or across the scaffold. A person skilled in the art will recognize that other perfusion culture conditions known in the art may be used for the cells described herein.

[0111] cell aggregates

[0112] In one other aspect, the formulation of the present disclosure comprises cell aggregates or spheroids. In one embodiment, the cell aggregate comprises the bioactive cell population described herein. In another embodiment, the cell aggregate comprises bioactive kidney cells, e.g., a combination of a kidney cell mixture, a concentrated kidney cell population, and a kidney cell fraction.

[0113] In certain embodiments, the bioactive kidney cells of the present disclosure may be cultured in a 3D format as further described herein. In some embodiments, the term “organoid” refers to an accumulation of cells, wherein the phenotype and / or function correspond to the intrinsic kidney. In some embodiments, the organoid comprises a mixed population of cells of various lineages typically found in vivo in a given tissue. In some embodiments, the organoid of the present disclosure is formed in vitro by any means, wherein the cells of the present disclosure form aggregates, which may consequently form spheroids, organoids, or a combination thereof. In some embodiments, such aggregates, spheroids, or organoids assume a structure corresponding to a specific organ. In some embodiments, such aggregates, spheroids, or organoids express a surface marker, which is typically expressed by cells of a specific organ. In some embodiments, such aggregates, spheroids, or organoids produce a compound or substance that is typically expressed by cells of a specific organ. In a specific embodiment, the cells of the present disclosure may be cultured on a natural substrate, such as gelatin. In another embodiment, the cells of the present disclosure may be cultured on a synthetic substrate, such as PGLA.

[0114] inactive cell population

[0115] As disclosed herein, a specific subfraction of a heterogeneous population of kidney cells, in which the bioactive component is concentrated and the inactive or undesirable component is depleted, provides therapeutic and regenerative results superior to the initial population. In a preferred embodiment, the formulation provided in this disclosure contains a cell population in which the B1 and / or B5 cell population is depleted. For example, B1 and / or B5 may be depleted in the following: a mixture of two or more of B2, B3, and B4 (or B4'); or a concentrated cell population of B2, B3, and B4 (or B4').

[0116] The B1 cell population consists of large granular cells of the collecting duct and tubular systems, where the cell population has a buoyancy density of less than about 1.045 g / m³. The B5 cell population has a buoyancy density greater than about 1.091 g / ml and consists of fragments and small cells of low particle size and viability.

[0117] Method of isolating and culturing a cell population

[0118] In one aspect, the formulation of the present disclosure comprises a cell population isolated from and / or cultured from kidney tissue. Methods for isolating and isolating kidney cell components, such as concentrated cell populations, to be used in formulations for therapeutic uses, including the treatment of kidney disease, anemia, EPO deficiency, tubular transport deficiency, and glomerular filtration deficiency are provided herein. In one embodiment, the cell population is isolated from newly degraded, i.e. mechanically or enzymatically degraded, kidney tissue or from a heterogeneous in vitro culture of mammalian kidney cells.

[0119] It may contain a heterogeneous mixture of renal cells cultured under hypoxic culture conditions prior to isolation, providing improved cell distribution and composition in B4, including B4', and B2 and / or B3 fractions. Oxygen-dependent cell enrichment was observed in B2 to B4 for renal cells isolated from diseased and disease-free renal cells. Without limitation to theory, this may be due to one or more of the following phenomena: 1) selective survival, death, or proliferation of specific cellular components during hypoxic culture periods; 2) changes in cell particle size and / or size in response to hypoxic culture, thereby affecting changes in buoyancy density and subsequent localization during density gradient separation; and 3) changes in cellular gene / protein expression in response to hypoxic culture periods, thereby resulting in differential cell characteristics within any given fraction of the gradient. Thus, in one embodiment, a cell population enriched with tubular cells, e.g., a preparation containing B2, is hypoxic-resistant.

[0120] Exemplary techniques for separating and isolating cell populations include separating on a density gradient based on different specific gravity of different cell types contained within the population of interest. The specific gravity of any given cell type may be influenced by intracellular particle size, intracellular water volume, and other factors. In one aspect, the present disclosure provides optimal gradient conditions for the isolation of B2 and B4, including B4', across a number of species including but not limited to humans, dogs, and rodents. In a preferred embodiment, the density gradient is used to obtain a novel concentrated population of tubular cell fractions, namely the B2 cell population, derived from a dichotomous population of kidney cells. In one embodiment, the density gradient is used to obtain a novel concentrated population of EPO-producing cell fractions, namely the B4 cell population, derived from a dichotomous population of kidney cells. In another embodiment, a density gradient is used to obtain concentrated subpopulations of renal tubular cells, glomerular cells, and endothelial cells. In one embodiment, EPO-producing and tubular cells are both separated from red blood cells and cell debris. In one embodiment, EPO-producing, glomerular, and vascular cells are separated from other cell types and from red blood cells and cell debris, and simultaneously, subpopulations of tubular cells and collecting duct cells are incidentally separated from other cell types and from red blood cells and cell debris. In one other embodiment, endocrine, glomerular, and / or vascular cells are separated from other cell types and from red blood cells and cell debris, and simultaneously, subpopulations of tubular cells and collecting duct cells are incidentally separated from other cell types and from red blood cells and cell debris.

[0121] In one aspect, the formulation of the present disclosure, based on the specific key features described below, comprises an OPTIPREP compound iodixanol treated with 60% nonionic iodine in water. ®(Axis-Shield) contains a cell population resulting from the partial use of a density gradient medium. However, a person skilled in the art will recognize that any density gradient or other means, such as immunological separation, utilizing cell surface markers known in the relevant part, which include the essential features for separating the cell population of the present disclosure, may be used. Furthermore, a person skilled in the art should recognize that the same cell features (size and particle size) that contribute to the separation of cell subpopulations through a density gradient may be used to separate cell subpopulations through flow cytometry (forward scatter = reflection of size through flow cytometry, and lateral scatter = reflection of particle size). Importantly, the density gradient medium must have low toxicity to the specific cell of interest. While the density gradient medium must have low toxicity to the specific cell of interest, the present disclosure considers the use of the gradient medium in a screening process for the cell of interest. Without being limited to theory, because there is significant cell damage between the loading and recovery steps, the cell population disclosed herein, recovered by a gradient containing iodixanol, appears to be iodixanol-resistant, suggesting that exposure to iodixanol under gradient conditions results in the elimination of specific cells. Cells appearing in specific bands after the iodixanol gradient are resistant to any adverse effects of iodixanol and / or density gradient exposure. Accordingly, the use of an additional control medium, which is a weak to moderate nephrotoxic, is also considered in the isolation and / or screening of the cell population for the formulations described herein. Additionally, the density gradient medium must not bind to proteins in human plasma or have an adverse effect on the primary function of the cells of interest.

[0122] In another aspect, the present disclosure provides a formulation containing a cell population in which a kidney cell type is enriched and / or depleted using fluorescence-activated cell sorting (FACS). In one embodiment, the kidney cell type is BD FACSAria™ Alternatively, it can be concentrated and / or depleted using an equivalent.

[0123] In another aspect, the formulation contains a cell population in which kidney cell types are enriched and / or depleted using self-cell sorting. In one embodiment, the kidney cell type is Miltenyi autoMACS ® It can be concentrated and / or depleted using a system or equivalent.

[0124] In another aspect, the formulation may comprise a three-dimensionally cultured renal cell population. In one aspect, the method of culturing the cell population is carried out via continuous perfusion. In one embodiment, a cell population cultured via three-dimensional culture and continuous perfusion exhibits greater cellularity and interconnectivity compared to a statically cultured cell population. In another embodiment, a cell population cultured via three-dimensional culture and continuous perfusion exhibits greater EPO expression and enhanced expression of renal tubule-associated genes, such as e-cadherin, compared to static culture of such cell populations. In another embodiment, a cell population cultured via continuous perfusion exhibits higher levels of glucose and glutamine consumption compared to a statically cultured cell population.

[0125] As described herein, low or hypoxic conditions may be used in the method for preparing a cell population for the formulation provided herein. However, the method for preparing the cell population may be used without the step of low oxygen conditioning. In one embodiment, standard oxygen conditions may be used.

[0126] A person skilled in the art will recognize that other methods of isolation and culture known in the art may be used for the cells described herein.

[0127] 3. Biomaterials

[0128] Various biomaterials may be combined with an activator to provide the therapeutic formulations of the present disclosure. The biomaterials may be in any suitable shape (e.g., beads) or form (e.g., liquid, gel, etc.). Bertram et al As described in U.S. Publication Application 20070276507 (incorporated herein by reference in its entirety), a polymer matrix or scaffold may be formed into a number of preferred arrangements to satisfy a number of overall system, geometric, or spatial constraints. In one embodiment, the matrix or scaffold of the present disclosure is three-dimensional and may be formed to fit the size and shape of an organ or tissue structure. For example, in the use of a polymer scaffold to treat kidney disease, anemia, EPO deficiency, tubular transport deficiency, or glomerular filtration deficiency, a three-dimensional (3-D) matrix may be used. Various differently formed 3-D scaffolds may be used. Naturally, the polymer matrix may be formed into different sizes and shapes to fit patients of different physiques. Additionally, the polymer matrix may be formed in other ways to accommodate the specific needs of the patient. In another embodiment, the polymer matrix or scaffold may be a biocompatible porous polymer scaffold. The scaffold is open-cell polylactic acid (OPLA ®), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenol, poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyether, polyether ether ketone, polyetherimide, polyether ketone, polyether sulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea-formaldehyde collagen, gelatin, alginate, laminin, It may be formed from various synthetic or naturally occurring materials, including but not limited to fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or copolymers or physical blends thereof. The scaffolding batch may vary from a liquid suspension to a soft porous scaffold or a porous scaffold with a rigid shape. In one embodiment, the batch is a solution in liquid form that may be a hydrogel.

[0129] Hydrogels can be formed from various polymeric materials and are useful in various biomedical applications. Hydrogels can be physically described as a three-dimensional network of hydrophilic polymers. Depending on the type of hydrogel, they contain varying percentages of water, although they are not soluble in water. Despite their high water content, hydrogels can bind large volumes of liquid due to the presence of hydrophilic residues. Hydrogels swell significantly without altering their gelatinous structure. The basic physical characteristics of hydrogels can be specifically modified depending on the properties of the polymers used and additional specific mechanisms for the product.

[0130] Preferably, the hydrogel is composed of a polymer, a biologically derived material, a synthetically derived material, or a combination thereof that is biologically inactive and physiologically compatible with mammalian tissues. The hydrogel material preferably does not induce an inflammatory response. Examples of other materials that may be used to form the hydrogel include (a) modified alginates, (b) polysaccharides that gel upon exposure to monovalent cations (i.e., gellan gum and carrageenan), (c) polysaccharides that are highly viscous liquids or thixotropic and form a gel over time through slow structural development (e.g., hyaluronic acid), (d) gelatin or collagen, and (e) polymer hydrogel precursors (e.g., polyethylene oxide-polypropylene glycol block copolymers and proteins). U.S. Patent No. 6,224,893 B1 provides a detailed description of various polymers suitable for making hydrogels and the chemical properties of such polymers.

[0131] The scaffolding or biomaterial feature may provide porous spaces in which cells can attach to and interact with the scaffolding or biomaterial material, and / or be trapped. In one embodiment, the porous scaffold or biomaterial allows for the addition or deposition of one or more populations or mixtures of cells on the biomaterial designed as a porous scaffold (e.g., by attachment of cells) and / or within the pores of the scaffold (e.g., by trapping of cells). In another embodiment, the scaffold or biomaterial allows or facilitates cell-to-cell and / or cell-to-biomaterial interactions within the scaffold to form structures as described herein.

[0132] In one embodiment, the biomaterial is 5.1 kDA to >2 x 10 6It consists of hyaluronic acid (HA) in the form of a hydrogel containing HA molecules having a size range of kDa. In another embodiment, the biomaterial is 5.1 kDa to >2 x 10 6 It consists of hyaluronic acid in the form of a porous foam, also containing HA molecules having a size range of kDa. In another embodiment, the biomaterial consists of a poly-lactic acid (PLA)-based foam having a pore size of about 50 microns to about 300 microns and an open-cell structure. In another embodiment, a specific cell population, preferably B2 and B4, directly provides and / or stimulates the synthesis of high molecular weight hyaluronic acid via hyaluronic acid synthase-2 (HAS-2), particularly after renal transplantation.

[0133] Biomaterials as described herein may also be designed or modified to respond to specific external conditions, e.g., in vitro or in vivo. In one embodiment, the biomaterial is temperature-sensitive (e.g., in vitro or in vivo). In another embodiment, the biomaterial is modified to respond to exposure to enzymatic degradation (e.g., in vitro or in vivo). The response of the biomaterial to external conditions may be fine-tuned as described herein. The temperature sensitivity of the described formulation may be altered by adjusting the percentage of the biomaterial within the formulation. For example, the percentage of gelatin in solution may be adjusted to control the temperature sensitivity of the gelatin in the final formulation (e.g., liquid, gel, beads, etc.). Alternatively, the biomaterial may be chemically crosslinked to provide greater resistance to enzymatic degradation. For example, a carbodiimide crosslinking agent may be used to chemically crosslink gelatin beads, thereby providing reduced sensitivity to endogenous enzymes.

[0134] In one aspect, the response of biomaterials to external conditions concerns the loss of structural integrity. Although temperature sensitivity and resistance to enzymatic degradation are provided above, there exist other mechanisms by which the loss of material integrity may occur in different biomaterials. These mechanisms are thermodynamic ( chamberlain Phase transitions such as dissolution and diffusion ( chamberlain , diffusion of ionic crosslinking agents from biomaterials into surrounding tissues)), chemical, enzymatic, pH ( chamberlain , pH-sensitive liposomes), ultrasound, and photodegradation (light transmission) may include, but are not limited to. The exact mechanism by which the biomaterial loses structural integrity will vary, but typically the mechanism is triggered at the time of implantation or post-implantation.

[0135] A person skilled in the art will recognize that other types of synthetic or naturally occurring materials known in the art may be used to form scaffolds as described herein.

[0136] In one aspect, a structure as described in this specification is made from the scaffold or biomaterial mentioned above.

[0137] 4. Structures

[0138] In one aspect, the present disclosure provides a formulation containing an implantable structure having one or more cell populations described herein for treatment in subjects requiring treatment for renal disease, anemia, or EPO deficiency. In one embodiment, the structure comprises a biocompatible material or biomaterial composed of one or more synthetic or naturally occurring biocompatible materials, a scaffold or matrix, and one or more cell populations or mixtures of cells described herein that are deposited on or embedded within the surface of the scaffold by attachment and / or capture. In a specific embodiment, the structure comprises one or more cell populations or mixtures of cells described herein that are coated with biomaterial and biomaterial component(s), deposited on a biomaterial component, deposited within a biomaterial component, attached to a biomaterial component, captured within a biomaterial component, embedded within a biomaterial component, inoculated with a biomaterial component, or combined with a biomaterial component. Any cell population described herein, including concentrated cell populations or mixtures thereof, may be used in combination with the matrix to form the structure.

[0139] In one aspect, the formulation contains a structure composed of biomaterials designed or modified to respond to external conditions as described herein. As a result, the nature of the association between the biomaterial and the cell population in the structure will change depending on the external conditions. For example, the association between a temperature-sensitive biomaterial and a cell population changes with temperature. In one embodiment, the structure contains a cell population and biomaterial that are in a substantially solid state at about 8°C or less and in a substantially liquid state at about ambient temperature or higher, wherein the cell population is suspended in the biomaterial at about 8°C or less.

[0140] However, cell populations are substantially free to move throughout the entire volume of the biomaterial at temperatures above ambient temperature. Suspending cell populations in a substantially solid state at lower temperatures provides stability advantages for cells, such as anchorage-dependent cells, compared to cells in fluid. Furthermore, suspending cells in a substantially solid state provides one or more of the following advantages: i) preventing cell sedimentation; ii) allowing cells to remain anchored to the biomaterial in a suspended state; iii) allowing cells to remain more uniformly dispersed throughout the entire volume of the biomaterial; iv) preventing the formation of cell aggregates; and v) providing better protection for cells during the storage and transport of the formulation. A formulation capable of possessing these features is advantageous for inducing administration to a subject, at least because the overall health of the cells in the formulation will be better and more uniform, and a constant dose of cells will be administered.

[0141] In another embodiment, the cell population or cell component to which the structure is deposited is a first renal cell population enriched with oxygen-tunable EPO-producing cells. In another embodiment, the first renal cell population contains glomerular and vascular cells in addition to oxygen-tunable EPO-producing cells. In one embodiment, the first renal cell population is a B4' cell population. In one other embodiment, the cell population or cell component(s) to which the structure is deposited comprises both the first enriched renal cell population and the second renal cell population. In some embodiments, the second cell population is not enriched with oxygen-tunable EPO-producing cells. In another embodiment, the second cell population is enriched with renal tubular cells. In another embodiment, the second cell population is enriched with renal tubular cells and contains collecting duct epithelial cells. In other embodiments, renal tubular cells may include, but are not limited to, the expression of one or more tubular cell markers including megalin, curbilin, hyaluronic acid synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, member RAS oncogene family (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9 (sodium / hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3 (Aldh1a3), and calpain-8 (Capn8). It is characterized.

[0142] In one embodiment, a cell population deposited on or combined with biomaterial or scaffold to form a structure is derived from various sources, such as autogenous sources. Additionally, non-autogenous sources, including but not limited to homogenous or homogeneous (autogenotype or homogenotype) sources, are also suitable for use.

[0143] A person skilled in the art will understand that there are various suitable methods for depositing cell populations into biomaterial to form structures, or for combining biomaterial with cell populations.

[0144] In one aspect, the structure is suitable for use in the method of use described herein. In one embodiment, the structure is suitable for administration to a subject requiring treatment for renal disease of any etiology, anemia, or EPO deficiency of any etiology. In another embodiment, the structure is suitable for administration to a subject requiring improvement and restoration of red blood cell homeostasis. In yet another embodiment, the structure is suitable for administration to a subject requiring improved renal function.

[0145] In another aspect, the present disclosure provides a structure for implantation into a subject requiring improved renal function, said structure comprising: a) a biomaterial comprising one or more biocompatible synthetic polymers or naturally occurring proteins or peptides; and b) a mixture of mammalian renal cells derived from a subject having renal disease comprising a first cell population, B2, comprising an isolated, concentrated population of tubular cells having a density of 1.045 g / mL to 1.052 g / mL, which is coated with the biomaterial, deposited on the biomaterial, or captured in the biomaterial, suspended in the biomaterial, embedded in the biomaterial, and / or otherwise combined with the biomaterial, and a second cell population, B4', comprising erythropoietin (EPO)-producing cells and vascular cells having a density of 1.063 g / mL to 1.091 g / mL but depleted of glomerular cells. In a specific embodiment, the mixture does not contain a B1 cell population including large granule cells of the collecting duct and tubular system having a density of < 1.045 g / ml, or a B5 cell population including fragments and small cells of low particle size and viability having a density of > 1.091 g / ml.

[0146] In one embodiment, the structure comprises a B4' cell population characterized by the expression of a vascular marker. In some embodiments, the B4' cell population is not characterized by the expression of a glomerular marker. In certain embodiments, the mixture may exhibit oxygen-tunable erythropoietin (EPO) expression. In all embodiments, the mixture may be derived from mammalian kidney tissue or cultured kidney cells.

[0147] In one embodiment, the structure comprises a biomaterial composed of a three-dimensional (3-D) porous biomaterial suitable for the capture and / or attachment of a mixture. In another embodiment, the structure comprises a biomaterial composed of a liquid or semi-liquid gel suitable for embedding, attaching, suspending, or coating mammalian cells. In another embodiment, the structure comprises a biomaterial composed primarily of a high molecular weight hyaluronic acid (HA) species in the form of a hydrogel. In another embodiment, the structure comprises a biomaterial composed primarily of a high molecular weight hyaluronic acid species in the form of a porous foam. In another embodiment, the structure comprises a biomaterial composed of a poly-lactic acid-based foam having pores of about 50 microns to about 300 microns. In another embodiment, the structure comprises one or more cell populations that may be derived from an autologous kidney sample for a subject requiring improved kidney function. In a specific embodiment, the sample is a kidney biopsy. In some embodiments, the subject has kidney disease. In other embodiments, the cell population is derived from a non-autologous kidney sample. In one embodiment, the structure provides erythrocyte homeostasis.

[0148] 5. Phenotypic Characterization of Kidney Cells

[0149] Cells isolated at any stage of the process can be characterized by their phenotype. In one embodiment, the cells are a concentrated heterogeneous renal cell population. In an additional embodiment, the concentrated heterogeneous renal cell population was cultured under hypoxic conditions for at least 24 hours. In an additional embodiment, a density gradient was formed in the concentrated heterogeneous renal cell population.

[0150] The presence (e.g., expression) and / or levels / quantities of various biomarkers in samples can be analyzed by numerous methodologies, including immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescently activated cell sorting ("FACS"), MassArray, proteomics, biochemical enzyme activity assays, and in situ ( in situ A multitude of methodologies are known in the art and understood by those skilled in the art, including but not limited to any one of the very diverse assays that can be performed by polymerase chain reaction ("PCR"), RNA-Seq, FISH, microarray analysis, gene expression profiling, and / or serial analysis of gene expression ("SAGE"), as well as other amplification type detection methods such as hybridization, Southern analysis, Northern analysis, whole-genome sequencing, quantitative real-time PCR ("qRT-PCR"), and branched DNA, SISBA, TMA, etc., and protein, gene, and / or tissue array analysis. Typical protocols for evaluating the status of genes and gene products are, for example, Ausubel et al This is revealed in ., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Combined immunoassays, such as those available from Rules Based Medicine or Meso Scale Discovery, may also be used.

[0151] In one aspect, a method for detecting the presence of two or more biomarkers in a heterogeneous kidney cell sample is provided, the method comprising the steps of contacting a sample with an antibody targeting a biomarker under conditions allowing binding of the antibody to its homologous ligand (i.e., the biomarker), and detecting the presence of the bound antibody by detecting, for example, whether a complex is formed between the antibody and the biomarker. In some embodiments, the detection of the presence of one or more biomarkers is by immunohistochemistry.

[0152] In specific embodiments, any antibody provided herein is useful for detecting the presence of a biomarker in a heterogeneous kidney cell sample. The term "detection" as used herein encompasses quantitative or qualitative detection. In specific embodiments, the biological sample comprises an SRC sample.

[0153] In a specific embodiment, heterogeneous renal cells are AQP1, AQP2, AQP4, calvidin, calponin, CD117, CD133, CD146, CD24, CD31 (PECAM-1), CD54 (ICAM-1), CD73, CK18, CK19, CK40 to 67, CK7, CK8, CK8 / 18, CK8 / 18 / 19, connexin 43, curbilin, CXCR4 (fusin), DBA, E-cadherin (CD324), EPO (erythropoietin), GGT1, GLEPP1 (glomerular epithelial protein 1), haptoglobulin, Itgb1 (integrin β1), KIM-1 / TIM-1 (renal injury molecule-1 / T-cell immunoglobulin and mucin-containing molecule), MAP-2 It is identified with one or more reagents that allow the detection of selected biomarkers from (microtubule-associated protein 2), megalin, N-cadherin, nephrine, NKCC (Na-K-Cl- cotransporter), OAT-1 (organic anion transporter 1), osteopontin, pan-cadherin, PCLP1 (podocalcin-like molecule 1), dodocin, SMA (smooth muscle alpha-actin), synaptopodin, THP (tam-horsefal protein), vimentin, and αGST-1 (alpha-glutathione S-transferase). In certain embodiments, the biomarker is detected by a monoclonal or polyclonal antibody.

[0154] In one embodiment, the detectable label contains a radioactive atom to form a radioconjugate. Various radioisotopes are available for the creation of the radioconjugate. Examples include 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 It includes radioisotopes of Pb and Lu. When a radioconjugate is used for detection, it is a radioactive atom for scintigraph research, e.g. 99 Tc-m (metastable nuclear isomer) or123 I, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), may include, for example, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0155] When more than one detectable label (including dyes) is used in a single test, it is desirable that the detectable labels be selected so that each label can be detected independently without substantial interference with any other detectable signal present in the sample. For example, the detectable labels (including dyes) may be different fluorescent molecules that exhibit different colors under detection conditions.

[0156] Detection can be performed by any suitable method, for example, based on immunofluorescence microscopy, flow cytometry, optical fiber scanning cytometry, or laser scanning cytometry.

[0157] In some embodiments, the expression of an intracellular biomarker is determined by evaluating mRNA in the cell. Methods for evaluating mRNA in cells are well known and include, for example, hybridization assays using complementary DNA probes (e.g., in situ hybridization using riboprobes labeled for one or more genes, Northern blot, and related techniques) and various nucleic acid amplification assays (e.g., RT-PCR using primers specific for one or more genes, and other amplification type detection methods, e.g., branched DNA, SISBA, TMA, etc.). In some embodiments, the expression of a biomarker in a test sample is compared with a reference sample. For example, the test sample may be a diseased tissue sample and the reference sample may be from normal tissue.

[0158] Samples from mammals can be conveniently analyzed for mRNA using Northern, dot blot, or PCR analysis. Additionally, these methods may include one or more steps that enable the determination of the level of target mRNA in the biological sample (e.g., by simultaneously examining the levels of comparative control mRNA sequences of "housekeeping" genes, such as actin family members). Optionally, the sequence of the amplified target cDNA can be determined.

[0159] Selective methods include protocols for testing or detecting mRNA, such as target mRNA, within tissues or samples using microarray technology. Using nucleic acid microarrays, test and control mRNA samples from test and control samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured so that the sequence and location of each member of the array are known. For example, selection of genes associated with a cell population whose expression can induce a regenerative response may be arrayed on a solid support. Hybridization of a labeled probe with a specific array member indicates that the sample from which the probe originated expresses that gene.

[0160] According to some embodiments, presence and / or level / amount is measured by observing the protein expression level of the aforementioned gene. In a specific embodiment, the method comprises the steps of contacting a biological sample with an antibody against the biomarker described herein under conditions allowing binding of the biomarker, and detecting whether a complex is formed between the antibody and the biomarker.

[0161] In specific embodiments, the presence and / or level / amount of a biomarker protein in a sample is tested using an IHC and staining protocol. IHC staining of cells has demonstrated that the method for determining or detecting the presence of proteins in a sample is a reliable method. In one aspect, the level of a biomarker is determined using a method comprising: (a) performing an IHC analysis of a sample (e.g., kidney cells) with an antibody; and (b) determining the level of the biomarker in the sample. In some embodiments, the IHC staining intensity is determined by comparison with a reference value.

[0162] IHC can be performed in combination with additional techniques, such as morphological staining and / or fluorescence in situ hybridization. Two general IHC methods are available: direct and indirect assays. According to the first assay, the binding of the target antigen to the antibody is determined directly. This direct assay utilizes labeled reagents, such as fluorescent tags or enzyme-labeled primary antibodies, which can be visualized without additional antibody interactions. In a typical indirect assay, an unconjugated primary antibody binds to the antigen, and then a labeled secondary antibody binds to the primary antibody. If the secondary antibody is suitable for enzyme labeling, a chromogenic or fluorescent substrate is added to provide visualization of the antigen. Signal amplification occurs because various secondary antibodies can interact with different epitopes on the primary antibody.

[0163] Typically, primary and / or secondary antibodies used for IHC will be labeled with detectable moieties. Numerous labels are available that can generally be grouped within the following categories: (a) radioisotopes, e.g. 35 S, 14 C, 125 I, 3 H, and 131I; (b) colloidal gold particles; (c) a fluorescent label comprising, but not limited to, a rare earth chelate (europium chelate), Texas red, rhodamine, fluorescein, dansil, risamine, umbelliferone, phycocriterin, phycocyanin, or commercially available fluorescent groups such as SPECTRUM ORANGE7 and SPECTRUM GREEN7 and / or any one or more derivatives of the above; (d) various enzyme-substrate labels are available, and U.S. Patent No. 4,275,149 provides a review of some of these. Examples of enzyme labels include luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazindione, maleate dehydrogenase, urease, peroxidase, e.g., horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc.

[0164] Examples of enzyme-substrate combinations include, for example, hydrogen peroxidase and horseradish peroxidase (HRP) as substrates; para-nitrophenyl phosphate and alkali phosphatase (AP) as chromogenic substrates; and chromogenic substrates (e.g., p-nitrophenyl-β-D-galactosidase) or fluorescent substrates (e.g., 4-methylumbeliferyl-β-D-galactosidase) and β-D-galactosidase (β-D-Gal). For a general review of these, refer to U.S. Patent Nos. 4,275,149 and 4,318,980.

[0165] In an exemplary method, a sample may be contacted with an antibody specific to the biomarker under conditions sufficient to form an antibody-biomarker complex, and then the complex may be detected. The presence of the biomarker can be detected in a number of ways, such as Western blotting and ELISA procedures for assembling a wide variety of tissues and samples, including plasma or serum. A wide variety of immunoassay technologies utilizing these assay formats are available, refer, for example, to U.S. Patents No. 4,016,043, 4,424,279, and 4,018,653. These include non-competitive types of unilateral and bilateral or "sandwich" assays, and traditional competitive binding assays. These assays also involve the direct binding of a target biomarker to a labeled antibody.

[0166] The presence and / or level / amount of selected biomarkers in tissue or cell samples may also be tested in the manner of functional or activity-based assays. For example, if the biomarker is an enzyme, assays known in the art may be performed to determine or detect the presence of a given enzyme activity in a tissue or cell sample.

[0167] In specific embodiments, samples are standardized for variability between assay operations, differences in the amount of the assayed biomarker, and variability in the quality of the samples used. This standardization can be achieved by detecting and incorporating levels of specific standardization biomarkers, including well-known endemic genes such as ACTB. Alternatively, standardization may be based on the mean or median signal of all assayed genes or a large subset thereof (global standardization technique). On a gene-for-gene basis, the measured standardized amount of the target tumor mRNA or protein is compared to the amount found in the reference set. The standardized expression level for each mRNA or protein per tumor tested for each target may be expressed as a percentage of the expression level measured in the reference set. The presence and / or expression level / amount measured in the specific target sample to be tested will fall within a certain percentile within this range, which can be determined by methods well known in the field.

[0168] In specific embodiments, the cytokeratin is selected from CK8, CK18, CK19, and combinations thereof. In specific embodiments, the cytokeratin is CK8, CK18, CK19, CK8 / CK18, CK8 / CK19, CK18 / CK19, or CK8 / CK18 / CK19, where " / " indicates a combination of adjacent cytokeratins. In all embodiments, the cytokeratin has an expression level of about 80%, about 85%, about 90%, or more than about 95%.

[0169] In specific examples, GGT is GGT-1. In all specific examples, GGT has an expression level of about 10%, about 15%, about 18%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or more than about 60%.

[0170] 6. How to Use

[0171] In another aspect, the formulation of the present disclosure may be administered for the treatment of a disease. For example, bioactive cells may be administered to an organ as part of the formulation described herein. In one embodiment, the bioactive cells may be supplied from the organ to be administered or from a source other than the target organ.

[0172] In one aspect, the present disclosure provides a method for treating a subject requiring treatment for renal disease, anemia, or EPO deficiency with a preparation containing a renal cell population and a mixture of renal cells as described herein. In one embodiment, the method comprises the step of administering to the subject a preparation containing a composition comprising a first renal cell population enriched with EPO-producing cells. In another embodiment, the first cell population is enriched with EPO-producing cells, glomerular cells, and vascular cells. In one embodiment, the first renal cell population is a B4' cell population. In another embodiment, the composition may further comprise one or more renal cell populations. In one embodiment, the additional cell population is a second cell population in which EPO-producing cells are not enriched. In another embodiment, the additional cell population is a second cell population in which EPO-producing cells, glomerular cells, or vascular cells are not enriched. In another embodiment, the composition also comprises a population of kidney cells or a mixture of kidney cells that are deposited in a biomaterial, deposited on a biomaterial, embedded in a biomaterial, coated in a biomaterial, suspended in a biomaterial, or captured in a biomaterial to form an implantable structure as described herein for the treatment of a disease or disorder described herein. In one embodiment, the cell population may be used alone or in combination with other cells or biomaterials, such as hydrogels, porous scaffolds, or intrinsic or synthetic peptides or proteins, to stimulate regeneration in acute or chronic disease states.

[0173] In another aspect, effective treatment of renal disease in a subject by the method disclosed herein may be observed through various indicators of erythropoiesis and / or renal function. In one embodiment, indicators of erythropoiesis homeostasis include, but are not limited to, hematocrit (HCT), hemoglobin (HB), mean particulate hemoglobin (MCH), red blood cell count (RBC), reticulocyte count, reticulocyte %, mean particulate volume (MCV), and red blood cell distribution width (RDW). In one other embodiment, indicators of renal function include, but are not limited to, serum albumin, albumin to globulin ratio (A / G ratio), serum phosphorus, serum sodium, renal size (measurable by ultrasound), serum calcium, phosphorus:potassium ratio, serum potassium, proteinuria, urinary creatinine, serum creatinine, blood nitric urea (BUN), cholesterol levels, triglyceride levels, and glomerular filtration rate (GFR). Additionally, various indicators of general health and well-being include, but are not limited to, weight gain or loss, survival, blood pressure (mean systemic blood pressure, diastolic blood pressure, or systolic blood pressure), and physical endurance.

[0174] In another embodiment, effective treatment with a bioactive renal cell preparation is demonstrated by the stabilization of one or more renal functions. Stabilization of renal function is demonstrated by the observation of a change in an indicator in a subject treated by the method provided herein, compared to the same indicator in a subject not treated by the method of this specification. Alternatively, stabilization of renal function may be demonstrated by the observation of a change in an indicator in a subject treated by the method of this specification, compared to the same indicator in the same subject prior to treatment. The change in the first indicator may be an increase or a decrease in value. In one embodiment, the treatment provided by the present disclosure may include the stabilization of blood urea nitrogen (BUN) levels in a subject, wherein the BUN level observed in the subject is lower than that of a subject with a similar disease state not treated by the method of this disclosure. In one other embodiment, the treatment may include the stabilization of serum creatinine levels in a subject, wherein the serum creatinine level observed in the subject is lower than that of a subject with a similar disease state not treated by the method of this disclosure. In another embodiment, treatment may include stabilization of hematocrit (HCT) levels in the subject, wherein the HCT levels observed in the subject are higher than those of a subject with a similar disease state not treated by the method of the present disclosure. In another embodiment, treatment may include stabilization of red blood cell (RBC) levels in the subject, wherein the RBC levels observed in the subject are higher than those of a subject with a similar disease state not treated by the method of the present disclosure. A person skilled in the art will recognize that one or more additional indicators described herein or known in the art may be measured to determine effective treatment of renal disease in the subject.

[0175] In another aspect, the present disclosure relates to a preparation for use in a method of providing erythrocyte homeostasis to a subject. In one embodiment, the method comprises (a) a population of kidney cells, e.g., B2 or B4', or a mixture of kidney cells, e.g., as described herein. The method comprises the steps of: (a) administering to a subject a preparation containing B2 / B4' and / or B2 / B3, or an enriched kidney cell population; and (b) determining whether the level of an erythropoietic indicator in a biological sample from the subject differs from the level of an indicator of a control, wherein the difference in the level of the indicator indicates (i) that the subject is responsive to the administration step (a) or (ii) that erythropoietic homeostasis is present in the subject. In another embodiment, the method comprises the steps of: (a) administering to a subject a preparation containing a kidney cell population or a mixture of kidney cells as described herein; and (b) determining whether the level of an erythropoietic indicator in a biological sample from the subject differs from the level of an indicator of a control, wherein the difference in the level of the indicator indicates (i) that the subject is responsive to the administration step(s) or (ii) that erythropoietic homeostasis is present in the subject. In another embodiment, the method comprises the steps of: (a) providing a biomaterial or a biocompatible polymer scaffold; (b) forming an implantable structure by depositing a population of kidney cells or a mixture of kidney cells of the present disclosure onto or within a biomaterial or scaffold in a manner described herein; (c) preparing a preparation containing the structure; (d) implanting the structure into a subject; and (e) determining whether the level of an erythropoietic indicator in a biological sample from the subject differs from the level of an indicator of a control, wherein the difference in the level of the indicator indicates (i) that the subject is responsive to the administration step (a), or (ii) that the subject has erythropoietic homeostasis.

[0176] In another aspect, the present disclosure relates to a formulation for use in a method of providing the stabilization of renal function and the restoration of red blood cell homeostasis to a subject who requires such stabilization, said subject who has both renal function deficiency and anemia and / or EPO deficiency. In one embodiment, the method comprises the step of comprising a formulation containing a population of renal cells or a mixture of renal cells as described herein, wherein the renal cells contain at least one of the following cell types: tubular-derived cells, glomerular-derived cells, interstitial-derived cells, collecting duct-derived cells, stromal tissue-derived cells, or cells derived from vascular structures. In another embodiment, the population or mixture comprises both EPO-producing cells and tubular epithelial cells, wherein the tubular cells are identified by at least one of the following markers: megalin, curbilin, hyaluronic acid synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, member RAS oncogene family (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9 (sodium / hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3 (Aldh1a3), and Calpain-8 (Capn8). In this embodiment, treatment of the subject will be demonstrated by improvement in at least one indicator of renal function along with improvement in at least one indicator of erythropoiesis, compared to the indicator of the untreated subject or the subject's pre-treatment indicator.

[0177] In one aspect, the present disclosure provides a preparation for use in (i) treatment of renal disease, anemia, or EPO deficiency by administering a renal cell population enriched with EPO-producing cells as described herein, or a mixture of renal cells containing a cell population enriched with EPO-producing cells; (ii) stabilization of renal function, (iii) restoration of erythrocyte homeostasis, or (iv) any combination thereof, wherein the beneficial effect of administration is greater than that of administering a cell population not enriched with EPO-producing cells. In another embodiment, the enriched cell population provides improved levels of serum blood urea nitrogen (BUN). In another embodiment, the enriched cell population provides improved protein retention in serum. In another embodiment, the enriched cell population provides improved levels of serum cholesterol and / or triglycerides. In another embodiment, the enriched cell population provides improved levels of vitamin D. In one embodiment, the concentrated cell population provides an improved phosphorus:calcium ratio compared to the non-concentrated cell population. In another embodiment, the concentrated cell population provides an improved level of hemoglobin compared to the non-concentrated cell population. In an additional embodiment, the concentrated cell population provides an improved level of serum creatinine compared to the non-concentrated cell population. In another embodiment, the concentrated cell population provides an improved level of hematocrit compared to the non-concentrated cell population. In an additional embodiment, the concentrated cell population provides an improved level of red blood cell count (RBC#) compared to the non-concentrated cell population. In one embodiment, the improved level of hematocrit is restored to 95% of the normal healthy level. In an additional embodiment, the concentrated cell population provides an improved reticulocyte count compared to the non-concentrated cell population. In another embodiment, the concentrated cell population provides an improved reticulocyte percentage compared to the non-concentrated cell population.In another embodiment, the concentrated cell population provides an improved level of red blood cell volume distribution width (RDW) compared to the non-concentrated cell population. In yet another embodiment, the concentrated cell population provides an improved level of hemoglobin compared to the non-concentrated cell population. In yet another embodiment, the concentrated cell population provides a hematopoietic response in the bone marrow, so the bone marrow cytoplasm is nearly normal and the bone marrow:red blood cell ratio is also nearly normal.

[0178] In another aspect, the present disclosure provides a formulation for use in (i) treatment of renal disease, anemia, or EPO deficiency by administering a concentrated cell population; (ii) stabilization of renal function; (iii) restoration of erythrocyte homeostasis; or (iv) any combination thereof, wherein the beneficial effect of administering the renal cell population or mixture of renal cell populations described herein is characterized by improved erythrocyte homeostasis compared to the beneficial effect provided by administering recombinant EPO (rEPO). In one embodiment, when the population or mixture is administered to a subject in need thereof, it provides improved erythrocyte homeostasis (determined by hematocrit, hemoglobin, or RBC#) compared to the administration of recombinant EPO protein. In one embodiment, when the population or mixture is administered, it provides improved levels of hematocrit, RBC, or hemoglobin compared to recombinant EPO, which are lower or higher than the hematocrit in the control by about 10% or less. In additional embodiments, a single dose or delivery of the population or mixture, when administered, provides an improvement in erythrocyte homeostasis (determined by an increase in hematocrit, hemoglobin, or RBC#) in subjects treated for a period significantly exceeding the period during which a single dose or delivery of recombinant EPO protein provides an improvement in erythrocyte homeostasis. In another embodiment, when the population or mixture is administered at the doses described herein, hematocrit, hemoglobin, or RBC# does not exceed about 110% of normal levels in a matching healthy control. In additional embodiments, when the population or mixture is administered at the doses described herein, it provides superior erythrocyte homeostasis (determined by hematocrit, hemoglobin, or RBC#) compared to recombinant EPO protein delivered at the doses described herein.In another embodiment, recombinant EPO is delivered at a dose of about 100 IU / kg, about 200 IU / kg, about 300 IU / kg, about 400 IU / kg, or about 500 IU / kg. A person skilled in the art will recognize that other dosages of recombinant EPO known in the art may be suitable.

[0179] Another embodiment of the present disclosure relates to the use of at least one cell population comprising the concentrated cell population and a mixture thereof described herein, or an implantable structure described herein, or a preparation containing a secreted product as described herein, for the preparation of a drug for treatment in a subject requiring treatment of kidney disease, anemia, or EPO deficiency, for the provision of such treatment in a subject requiring erythrocyte homeostasis, for the improvement of such treatment in a subject requiring improvement of kidney function, or for the provision of a regenerative effect on the intrinsic kidney.

[0180] Another embodiment of the present disclosure relates to a formulation containing specific concentrated cell population(s) for treating renal disease of a specific etiology, based on the selection of specific cell subpopulation(s) based on specific proven therapeutic properties.

[0181] In another embodiment, the present disclosure provides a preparation for use in a method of treating a subject requiring treatment for kidney disease, the method comprising: administering to a subject a preparation comprising a mixture of mammalian kidney cells comprising a first cell population, B2, comprising an isolated, concentrated population of tubular cells having a density of 1.045 g / mL to 1.052 g / mL, and a second cell population, B4', comprising erythropoietin (EPO)-producing cells and vascular cells having a density of 1.063 g / mL to 1.091 g / mL, but depleted of glomerular cells, wherein the mixture does not comprise a B1 cell population comprising large granule cells of the collecting duct and tubular system having a density of < 1.045 g / mL, or a B5 cell population comprising fragments and small cells having low particle size and viability having a density of > 1.091 g / mL. In a specific embodiment, the method comprises the step of determining from a test sample from a subject that the level of a renal function indicator differs from the level of the indicator in a control, wherein the difference in the level of the indicator indicates a reduction, stabilization, or improvement of one or more renal function declines in the subject. In one embodiment, the B4' cell population used in the method is characterized by the expression of a vascular marker. In a specific embodiment, the B4' cell population used in the method is not characterized by the expression of a glomerular marker. In one embodiment, the mixture of cells used in the method may have oxygen-tunable erythropoietin (EPO) expression. In a specific embodiment, the renal disease to be treated by the method of the present disclosure involves erythropoietin (EPO) deficiency. In a specific embodiment, EPO deficiency is anemia. In some embodiments, EPO deficiency or anemia occurs secondarily to renal failure in the subject.In some embodiments, EPO deficiency or anemia occurs secondarily to a disease selected from the group consisting of chronic renal failure, primary EPO deficiency, chemotherapy or antiviral therapy, non-myeloid cancer, HIV infection, liver disease, heart failure, rheumatoid arthritis, or multiple organ system failure. In certain embodiments, the composition used in the present invention further comprises a biomaterial comprising one or more biocompatible synthetic polymers and / or naturally occurring proteins or peptides, wherein the mixture is coated with the biomaterial, deposited on the biomaterial, deposited within the biomaterial, captured within the biomaterial, suspended in the biomaterial, embedded within the biomaterial, and / or otherwise combined with the biomaterial. In certain embodiments, the mixture used in the formulation of the present disclosure is derived from mammalian kidney tissue or cultured mammalian kidney cells. In other embodiments, the mixture is derived from a kidney sample that is autologous to the subject requiring treatment. In one embodiment, the sample is a kidney biopsy. In another embodiment, the formulation contains a mixture derived from a non-autologous kidney sample.

[0182] In another aspect, the present disclosure provides for the use of a formulation containing the cell products and mixtures described herein or the implantable structures of the present disclosure for the preparation of a drug useful for treating kidney disease, anemia, or EPO deficiency in subjects requiring treatment.

[0183] In another aspect, the present disclosure provides a formulation for use in a method for regeneration of the intrinsic kidney in subjects requiring such regeneration. In one embodiment, the method comprises the step of administering or implanting the cell population, mixture, or structure described herein to the subject. The regenerated intrinsic kidney may be characterized by a number of indicators, including but not limited to the development of function or capacity in the intrinsic kidney, improvement of function or capacity in the intrinsic kidney, and expression of specific markers in the intrinsic kidney. In one embodiment, the developed or improved function or capacity may be observed based on various indicators of erythrocyte homeostasis and kidney function described above. In another embodiment, the regenerated kidney is characterized by differential expression of one or more stem cell markers. The stem cell markers may be one or more of the following: SRY (sex determining region Y)-box 2 (Sox2); undifferentiated embryonic cell transcription factor (UTF1); nodular homolog from mouse (NODAL); Prominin 1 (PROM1) or CD133 (CD133); CD24; any combination thereof (Ilagan which is incorporated herein by reference in its entirety) et al (See PCT / US2011 / 036347). In another embodiment, the expression of stem cell marker(s) is upregulated compared to the control.

[0184] The cell populations described herein, including concentrated cell populations and mixtures thereof, and structures containing such populations may be used to provide a regenerative effect on the native kidney. Such effect may be provided by the cells themselves and / or by products secreted from the cells. The regenerative effect may be characterized by one or more of the following: a reduction in epithelial-mesenchymal transition (which may be achieved through the attenuation of TGF-β signaling); a reduction in renal fibrosis; a reduction in renal inflammation; differential expression of stem cell markers in the native kidney; migration of transplanted cells and / or native cells to a site of renal injury, e.g., tubular injury; grafting of transplanted cells at a site of renal injury, e.g., tubular injury; stabilization of one or more indicators of renal function (as described herein); restoration of erythrocyte homeostasis (as described herein); and any combination thereof.

[0185] 7. How to monitor playback

[0186] In another aspect, the present disclosure provides a prognostic method for monitoring the regeneration of the intrinsic kidney after administration or transplantation of a preparation containing the cell population, mixture, or structure described herein to a subject. In one embodiment, the method comprises the step of detecting the expression level of a marker in a test sample and a control sample obtained from a subject, wherein a higher level of expression of the marker in the test sample compared to the control sample is a prognostic for the regeneration of the intrinsic kidney in the subject. In another embodiment, the method comprises detecting the expression of one or more stem cell markers in a sample. The stem cell markers may be selected from Sox2; UTF1; NODAL; CD133; CD24; and combinations thereof (Ilagan et al(Refer to Example 11 of PCT / US2011 / 036347). The detection step may include determining whether the expression of the stem cell marker(s) is upregulated or higher in the test sample compared to the control sample, wherein a higher level of expression prognoses the regeneration of the subject's intrinsic kidney. In one other embodiment, mRNA expression of the stem cell marker(s) is detected. In another embodiment, detection of mRNA expression may occur via a PCR-based method, e.g., qRT-PCR. Additionally, in situ hybridization may also be used for the detection of mRNA expression. In yet another embodiment, polypeptide expression of the stem cell marker may also be detected using an anti-stem cell marker agent. In one other embodiment, the agent is an antibody targeting the marker. In yet another embodiment, stem cell marker polypeptide expression is detected using immunohistochemistry or Western blot. A person skilled in the art will recognize other methods for detecting mRNA and / or polypeptide expression of markers.

[0187] In another aspect, the present disclosure provides a method for evaluating the prognosis of a patient after the transplantation or administration of a preparation containing the cell population, mixture, or structure described herein. In one embodiment, the method comprises the steps of: detecting a level of marker expression in a test sample obtained from said subject; (b) determining the level of expression in the test sample compared to the level of marker expression for a control sample (or control reference value); and (c) predicting the prognosis of regeneration of the patient based on the determination of the level of marker expression, wherein a higher level of marker expression in the test sample compared to the control sample (control reference value) predicts regeneration in the subject.

[0188] In one other aspect, the present disclosure provides a prognostic method for monitoring regeneration of an intrinsic kidney after administration or transplantation of a preparation containing the cell population, mixture, or structure described herein to a subject, wherein a non-invasive method is utilized. As an alternative to tissue biopsy, regeneration outcomes in the treated subject may be evaluated from body fluids, such as urine tests. It has been found that microvesicles obtained from a subject-derived urine source contain specific components, which are ultimately not limited to but include specific proteins and miRNAs derived from the kidney cell population affected by treatment with the cell population of the present disclosure. These components may include factors associated with stem cell replication and differentiation, apoptosis, inflammation, and immune regulation. Temporal analysis of microvesicle-associated miRNA / protein expression patterns allows for continuous monitoring of regeneration outcomes within the kidney of a subject who has received the cell population, mixture, or structure of the present disclosure.

[0189] These kidney-derived vesicles and / or the luminal contents of kidney-derived vesicles excreted in the subject's urine can be analyzed for biomarkers indicating regeneration results.

[0190] In one embodiment, the present disclosure provides a method for evaluating whether a patient with kidney disease (KD) responds to treatment with a therapeutic agent. The method may include the step of determining or detecting the amount of vesicles or their luminal contents in a test sample obtained from a KD patient treated with the therapeutic agent, by comparing or in comparison to the amount of vesicles in a control sample, wherein a higher or lower amount of vesicles or their luminal contents in the test sample compared to the amount of vesicles or their luminal contents in the control sample indicates the responsiveness of the treated patient to treatment with the therapeutic agent.

[0191] The present disclosure also provides a method for monitoring the efficacy of treatment with a therapeutic agent in KD patients. In one embodiment, the method comprises the step of determining or detecting the amount of vesicles in a test sample obtained from a KD patient treated with a therapeutic agent by comparing, or by comparison with, the amount of vesicles or their luminal contents in a control sample, wherein a higher or lower amount of vesicles or their luminal contents in the test sample compared with the amount of vesicles or their luminal contents in the control sample indicates the efficacy of treatment with a therapeutic agent in KD patients.

[0192] The present disclosure provides a method for identifying a patient subgroup in which an agent is effective for treating a disease of the heart (KD). In one embodiment, the method comprises the step of determining a correlation between the presence of a sample vesicle or its luminal contents from a patient subgroup and the efficacy of the agent, in comparison to a sample vesicle or its luminal contents obtained from a control sample, wherein a higher or lower amount of sample vesicles from the patient subgroup compared to a control sample vesicle or its luminal contents indicates that the agent is effective for treating KD in the patient subgroup.

[0193] The step of determining or detecting may include a step of analyzing the amount of miRNA or other secreted products that may be present in a test sample, such as urine.

[0194] A non-invasive prognostic method may include the step of obtaining a urine sample from a subject before and / or after administration or transplantation of the cell population, mixture, or structure described herein. Vesicles and other secretory products may be isolated from the urine sample using standard techniques, including but not limited to centrifugation to remove unwanted debris (Zhou et al . 2008. Kidney Int. 74(5):613-621; Skog et al. U.S. Published Patent Application No. 20110053157, each of which is incorporated herein by reference in its entirety).

[0195] The present disclosure relates to a non-invasive method for detecting regenerative outcomes in a subject after treatment. The method comprises detecting vesicles or their luminal contents in urine from a treated subject. The luminal contents may be one or more miRNAs. Detection of combinations or panels of individual miRNAs may be suitable for this prognostic method. Exemplary combinations include two or more of the following: miR-24; miR-195; miR-871; miR-30b-5p; miR-19b; miR-99a; miR-429; let-7f; miR-200a; miR-324-5p; miR-10a-5p; and any combination thereof. In one embodiment, the combination of miRNAs may include two, three, four, five, six, seven, eight, nine, ten, eleven or more individual miRNAs. A person skilled in the art will recognize that other miRNAs and combinations of miRNAs may be suitable for use in these prognostic methods. Additional sources of miRNAs include miRBase at http: / / mirbase.org, which is managed and maintained by the Department of Life Sciences at the University of Manchester.

[0196] A person skilled in the art will recognize that a prognostic method for detecting regeneration may be suitable for subjects treated with other therapeutic agents known in the art, apart from the cell populations and structures described herein.

[0197] In some embodiments, the determination step involves the use of a software program executed by a suitable processor for the purpose of (i) measuring the differential level of marker expression (or vesicles / vesicle contents) between a test sample and a control; and / or (ii) analyzing the data obtained from the step of measuring the differential level of marker expression between a test sample and a control. Suitable software and processors are well known in the art and are commercially available. The program is stored in software stored on an actual medium such as a CD-ROM, floppy disk, hard disk, DVD, or memory associated with the processor, but a person skilled in the art will readily recognize that the whole program or part thereof may be executed alternatively by a device other than the processor and / or stored in firmware and / or dedicated hardware in a well-known manner.

[0198] After the decision phase, measurement results, findings, diagnoses, predictions, and / or treatment recommendations are typically recorded and conveyed, for example, to technicians, physicians, and / or patients. In specific embodiments, a computer will be used to convey this information to interested parties, such as patients and / or attending physicians. In some embodiments, the assay may be performed or the results analyzed in a country or jurisdiction different from the country or jurisdiction to which the results or diagnosis are conveyed.

[0199] In a preferred embodiment, prognosis, prediction, and / or treatment recommendations based on levels of marker expression measured in subjects having differential levels of marker expression are delivered to the subject as soon as possible after the assay is completed and the prognosis and / or prediction are obtained. Results and / or related information may be delivered to the subject by the subject's attending physician. Alternatively, results may be delivered directly to the subject by any means of communication, including letters, electronic communication, such as email or telephone. As in the case of email delivery, delivery may be facilitated by the use of a computer. In a specific embodiment, information including the results of a prognostic test and / or conclusions derived therefrom and / or treatment recommendations based on the test may be generated and automatically delivered to the subject using a combination of computer hardware and software familiar to telecommunications technicians. One example of a healthcare-oriented communication system is described in U.S. Patent No. 6,283,761; however, the present disclosure is not limited to the method of utilizing this particular communication system. In a specific embodiment of the method of the present disclosure, all or some of the steps of the method, including assay of a sample, prognosis and / or prediction of regeneration, and delivery of assay results or prognosis, may be performed in various (e.g., overseas) jurisdictions.

[0200] In another aspect, the prognostic method described in this specification provides information to interested parties regarding the success of regeneration of transplantation or administration.

[0201] In all embodiments, the method of providing a regenerated kidney to a subject requiring such treatment as described herein may include a post-transplantation step of a prognostic evaluation of regeneration as described above.

[0202] 8. Bioactive cell preparations

[0203] The formulation described herein comprises a biomaterial having properties that form a desirable environment for an activator, e.g., a bioactive kidney cell, to be administered to a subject. In one embodiment, the formulation comprises a first biomaterial that provides a desirable environment from the time the activator is prepared as a biomaterial until the time it is administered to the subject. In one other embodiment, the desirable environment relates to the advantage of having the bioactive cell suspended in a substantially solid state compared to the cell in a fluid (as described herein) prior to administration to the subject. In another embodiment, the first biomaterial may be a temperature-sensitive biomaterial. The temperature-sensitive biomaterial may have (i) a substantially solid state at about 8°C or lower, and (ii) a substantially liquid state at ambient temperature or higher. In one embodiment, the ambient temperature is about room temperature.

[0204] In another aspect, the formulation contains a bioactive cell combined with a second biomaterial that provides a favorable environment for the combined cell from the time of manufacture until a time after administration to the subject. In one embodiment, the favorable environment provided by the second biomaterial relates to the advantage of administering the cell to the biomaterial that retains structural integrity up to the time of administration to the subject and for a time after administration. In one embodiment, structural integrity of the second biomaterial after transplantation takes minutes, hours, days, or weeks. In one embodiment, structural integrity takes less than one month, less than one week, less than one day, or less than one hour. Relatively short structural integrity provides a formulation capable of delivering the activator and biomaterial to a target site within the tissue or organ by controlled manipulation, placement, or dispersion without interference or inhibition of the interaction between the placed tissue or organ and the incorporated element.

[0205] In another embodiment, the second biomaterial is a temperature-sensitive biomaterial having a sensitivity that differs from that of the first biomaterial. The second biomaterial may have (i) a substantially solid state at or below about ambient temperature, and (ii) a substantially liquid state at or above about 37°C. In one embodiment, the ambient temperature is about room temperature.

[0206] In one embodiment, the second biomaterial is a cross-linked bead. The cross-linked bead may have an in vitro residence time that is finely adjustable depending on the degree of cross-linking, as described herein. In another embodiment, the cross-linked bead contains a bioactive cell and is resistant to enzymatic degradation as described herein.

[0207] The formulations of the present disclosure may comprise a first biomaterial combined with an activator, such as a bioactive cell, with or without a second biomaterial combined with an activator, such as a bioactive cell. If the formulation comprises a second biomaterial, it may be a temperature-sensitive bead and / or a cross-linked bead. Various representative formulations are provided in the following examples (see also FIG. 3-7).

[0208] The bioactive cell products, mixtures, and / or structures described herein may be administered as bioactive cell preparations. In one aspect, the preparation comprises a cell and one or more biomaterials that provide stability for the bioactive cell products, mixtures, and / or structures described herein. In one embodiment, the biomaterial is a temperature-sensitive biomaterial capable of maintaining at least two different phases or states depending on temperature. The biomaterial may maintain a first state at a first temperature, a second state at a second temperature, and / or a third state at a third temperature. The first, second, or third states may be substantially solid, substantially liquid, or substantially semi-solid or semi-liquid states. In one embodiment, the biomaterial has a first state at a first temperature and a second state at a second temperature, wherein the first temperature is lower than the second temperature.

[0209] In one other embodiment, the temperature-sensitive biomaterial is in a substantially solid state at a temperature of about 8°C or lower. In another embodiment, the substantially solid state is maintained at about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, or about 8°C. In one embodiment, the substantially solid state takes the form of a gel. In another embodiment, the state of the temperature-sensitive biomaterial is in a substantially liquid state above this temperature. In one embodiment, the substantially liquid state is maintained at about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, or about 37°C. In one embodiment, the ambient temperature is about room temperature.

[0210] In another embodiment, the state of the temperature-sensitive biomaterial is substantially solid at a temperature below about ambient temperature. In one embodiment, the ambient temperature is about room temperature. In another embodiment, the substantially solid state is maintained at about 17°C, about 16°C, about 15°C, about 14°C, about 13°C, about 12°C, about 11°C, about 10°C, about 9°C, about 8°C, about 7°C, about 6°C, about 5°C, about 4°C, about 3°C, about 2°C, or about 1°C. In one embodiment, the substantially solid state has the form of beads. In another embodiment, the state of the temperature-sensitive biomaterial is substantially liquid at a temperature above about 37°C. In one other embodiment, the substantially solid state is maintained at about 37°C, about 38°C, about 39°C, or about 40°C.

[0211] Temperature-sensitive biomaterials may be provided in the form of solution, in the form of beads, or in other suitable forms described herein and / or known to those skilled in the art. Cell populations and products described herein may be coated with the temperature-sensitive biomaterial, deposited on said biomaterial, embedded within said biomaterial, attached to said biomaterial, smeared on said biomaterial, suspended, or captured in said biomaterial. Alternatively, the temperature-sensitive biomaterial may be provided without any cells, for example, in the form of spacer beads.

[0212] In another embodiment, the temperature-sensitive biomaterial has a transition state in the first state and the second state. In one embodiment, the transition state is a solid-to-liquid transition state at a temperature of about 8°C to about ambient temperature. In one embodiment, the ambient temperature is about room temperature. In one other embodiment, the solid-to-liquid transition state occurs at one or more of the temperatures of about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, to about 18°C.

[0213] Temperature-sensitive biomaterials have a specific viscosity at a given temperature, measured in centipoise (cP). In one embodiment, the biomaterial has a viscosity of about 1 cP to about 5 cP, about 1.1 cP to about 4.5 cP, about 1.2 cP to about 4 cP, about 1.3 cP to about 3.5 cP, about 1.4 cP to about 3.5 cP, about 1.5 cP to about 3 cP, about 1.55 cP to about 2.5 cP, or about 1.6 cP to about 2 cP at 25°C. In another embodiment, a 0.75% (w / v) solution has a viscosity of about 1.0 cP to about 1.15 cP at 37°C. At 37°C, the viscosity may be about 1.0 cP, about 1.01 cP, about 1.02 cP, about 1.03 cP, about 1.04 cP, about 1.05 cP, about 1.06 cP, about 1.07 cP, about 1.08 cP, about 1.09 cP, about 1.10 cP, about 1.11 cP, about 1.12 cP, about 1.13 cP, about 1.14 cP, or about 1.15 cP. In one other embodiment, the biomaterial is a gelatin solution. Gelatin is present in the solution at about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, or about 1% (w / v). In one embodiment, the biomaterial is a 0.75% (w / v) gelatin solution in PBS. In one embodiment, the 0.75% (w / v) solution has a viscosity of about 1.6 cP to about 2 cP at 25°C. In one embodiment, the 0.75% (w / v) solution has a viscosity of about 1.07 cP to about 1.08 cP at 37°C. The gelatin solution may be provided in PBS, DMEM, or another suitable solvent.

[0214] In one aspect, the bioactive cell preparation also comprises a cell viability agent. In one embodiment, the cell viability agent is selected from the group consisting of antioxidants, oxygen carriers, immunomodulatory factors, cell recruitment factors, cell adhesion factors, anti-inflammatory agents, angiogenic factors, wound healing factors, and products secreted from bioactive cells.

[0215] Antioxidants are characterized by their ability to inhibit the oxidation of other molecules. Antioxidants are 6-hydroxy-2,5,7,8-tetramethylchloroman-2-carboxylic acid (Trolox ® ), carotenoids, flavonoids, isoflavones, ubiquinones, glutathione, lipoic acid, superoxide dismutase, ascorbic acid, vitamin E, vitamin A, mixed carotenoids (e.g., beta-carotene, alpha-carotene, gamma-carotene, lutein, lycopene, phytophene, phytofluene, and astaxanthin), selenium, coenzyme Q10, indole-3-carbinol, proanthocyanidins, resveratrol, quercetin, catechin, salicylic acid, curcumin, bilirubin, oxalic acid, phytic acid, lipoic acid, vanillic acid, polyphenols, ferulic acid, theaflavin, and one or more derivatives thereof. A person skilled in the art will recognize other suitable antioxidants for use in the present disclosure.

[0216] Oxygen carriers are characterized by their ability to transport and release oxygen. They include, but are not limited to, perfluorocarbons and pharmaceuticals containing perfluorocarbons. Suitable perfluorocarbon-based oxygen carriers include perfluorooctyl bromide (C8F17Br); perfluorodicolotan (C8F16C12); perfluorodecyl bromide; perfluoroobron; perfluorodecalin; perfluorotriphophylamine; perfluoromethylcyclopiperidine; and Fluosol ® (Perfluorodecalin & Perfluorotriphophylamine); Perftoran ® (Perfluorodecalin & Perfluoromethylcyclopiperidine); Oxygent ®(perfluorodecyl bromide & perfluorobron); Ocycyte™ (perfluoro(tert-butylcyclohexane)) is included, but not limited thereto. A person skilled in the art will recognize other suitable perfluorocarbon-based oxygen carriers for use in the present disclosure.

[0217] Immunomodulatory factors include, but are not limited to, osteopontin, FAS ligand factor, interleukin, transforming growth factor beta, serum-derived growth factor, clusterin, transferrin, secreted protein (RANTES) that is regulated upon action, expressed by normal T-cells, plasminogen activator inhibitor-1 (Pai-1), tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), alpha-1 microglobulin, and beta-2-microglobulin. A person skilled in the art will understand other suitable immunomodulatory factors for use in the present disclosure.

[0218] Anti-inflammatory agents or immunosuppressants (described below) may also be part of the formulation. A person skilled in the art will understand other suitable antioxidants for use in this formulation and / or treatment.

[0219] Cell recruitment factors include, but are not limited to, monocyte chemotactic protein 1 (MCP-1) and CXCL-1. A person skilled in the art will recognize other suitable cell recruitment factors for use in this formulation and / or therapy.

[0220] Cell adhesion factors include, but are not limited to, fibronectin, procollagen, collagen, ICAM-1, connective tissue growth factor, laminin, proteoglycans, and specific cell adhesion peptides such as RGD and YSIGR. A person skilled in the art will recognize other suitable cell adhesion factors for use in this formulation and / or therapy.

[0221] Angiogenic factors include, but are not limited to, matrix metalloprotease 1 (MMP1), matrix metalloprotease 2 (MMP2), vascular endothelial growth factor F (VEGF), matrix metalloprotease 9 (MMP-9), tissue inhibitors or metalloprotease-1 (TIMP-1), vascular endothelial growth factor F (VEGF), and angiopoietin-2 (ANG-2). A person skilled in the art will recognize other suitable angiogenic factors for use in this formulation and / or therapy.

[0222] Wound healing factors include, but are not limited to, keratinocyte growth factor 1 (KGF-1), tissue plasminogen activator (tPA), calvidin, clusterin, cysteine ​​C, and trepoil factor 3. A person skilled in the art will recognize other suitable wound healing factors for use in formulations and / or treatments.

[0223] The product secreted from the bioactive cell described in this specification may also be added to the bioactive cell preparation as a cell viability agent.

[0224] In one other aspect, the formulation comprises a population of temperature-sensitive biomaterials and biocompatible beads containing biomaterials as described herein. In one embodiment, the beads are crosslinked. The crosslinking is performed using any suitable crosslinking agent known to a person skilled in the art, e.g., carbodiimide; Aldehydes (e.g., furfural, acrolein, formaldehyde, glutaraldehyde, glycerylaldehyde), succinimide-based crosslinking agents {bis(sulfosuccinimidyl) suberate (BS3), disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), dithiobis(succinimidyl propionate), ethylene glycol bis(sulfosuccinimidyl succinate), ethylene glycol bis(succinimidyl succinate) (EGS), bis(sulfosuccinimidyl) glutarate (BS2G), disuccinimidyl tartrate (DST)}; epoxides (ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether); saccharides (glucose and aldoses sugars); sulfonic acids and p-toluenesulfonic acids; carbonyldiimidazole; This can be achieved using genipin; imine; ketone; diphenylphosphorylazide (DDPA); terephthaloyl chloride; cerium (III) nitrate hexahydrate; microbial transglutaminase; and hydrogen peroxide. A person skilled in the art will recognize other suitable crosslinking agents and crosslinking methods for use in this method, formulations and / or treatments.

[0225] In one embodiment, the beads are carbodiimide-crosslinked beads. The carbodiimide-crosslinked beads can be crosslinked with a carbodiimide selected from the group consisting of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), DCC-N,N'-dicyclohexylcarbodiimide (DCC), and N,N'-diisopropylcarbodiimide (DIPC). Beads treated with lower concentrations of EDC were predicted to have a greater number of free primary amines, while samples treated with high concentrations of crosslinking agent would have most of the primary amines involved in amide bonding. The intensity of the orange color developed by the covalent bond between the primary amine and picrylsulfonic acid, detectable by spectrophotometry at 335 nm, is proportional to the number of primary amines present in the sample. When normalized per milligram of protein present in the sample, an inverse correlation can be observed between the initial concentration of EDC used for crosslinking and the number of free amines present. This result indicates differential bead crosslinking, which is governed by the amount of carbodiimide used in the reaction. Generally, crosslinked beads exhibit a reduced number of free primary amines compared to non-crosslinked beads. The number of free primary amines can be detected by spectrophotometry at approximately 335 nm.

[0226] Cross-linked beads have reduced susceptibility to enzymatic degradation compared to non-cross-linked biocompatible beads, and thus provide beads with finely tuned in vivo residence times. For example, cross-linked beads are resistant to endogenous enzymes, such as collagenase. The delivery of cross-linked beads is part of a delivery system focused on the development and generation of biomaterials that promote one or more of the following: (a) delivery of cells attached to a desired site and formation of space for the regeneration and endogeneity of progenitor tissue and vascular supply; (b) ability to persist at a site long enough for cells to establish, function, remodel, and secrete their own extracellular matrix (ECM); (c) enhancement of the integration of transplanted cells with surrounding tissues; (d) ability to transplant cells in a substantially solid form; (e) short-term structural integrity that does not provide significant barriers to the integration of delivered cells / materials with host tissues or tissue endogeneity; and (f) localization of in vivo delivery in a substantially solid form to prevent cell dispersal into tissues during transplantation. (g) improved stability and viability of anchorage-dependent cells compared to cells suspended in fluid; and (h) biphasic release profile when cells are delivered in i) a substantially solid form (e.g., attached to beads) and ii) a substantially liquid form (e.g., suspended in fluid).

[0227] In one embodiment, the present disclosure provides cross-linked beads containing gelatin. Non-cross-linked gelatin beads are not suitable for bioactive cell preparations because they rapidly lose integrity and cells are destroyed at the injection site. In contrast, highly cross-linked gelatin beads can persist at the injection site for a very long time and new ( de-novo) It may interfere with ECM secretion, cell integration, and tissue regeneration. The present disclosure allows for the in vivo residence time of crosslinked beads to be finely tuned. While different concentrations of crosslinking agent of carbodiimide were used to match the biodegradability of the biomaterial, overall reaction conditions were maintained for all samples. For example, the enzymatic susceptibility of carbodiimide-crosslinked beads can be finely tuned by varying the concentration of the crosslinking agent from about 0 to about 1 M. In some embodiments, the concentration is about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about The concentration is 45 mM, approximately 46 mM, approximately 47 mM, approximately 48 mM, approximately 49 mM, approximately 50 mM, approximately 55 mM, approximately 60 mM, approximately 65 mM, approximately 70 mM, approximately 75 mM, approximately 80 mM, approximately 85 mM, approximately 90 mM, approximately 95 mM, or approximately 100 mM. The crosslinker concentration may also be approximately 0.15 M, approximately 0.2 M, approximately 0.25 M, approximately 0.3 M, approximately 0.35 M, approximately 0.4 M, approximately 0.45 M, approximately 0.5 M, approximately 0.55 M, approximately 0.6 M, approximately 0.65 M, approximately 0.7 M, approximately 0.75 M, approximately 0.8 M, approximately 0.85 M, approximately 0.9 M, approximately 0.95 M, or approximately 1 M.In another embodiment, the crosslinking agent is 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). In one embodiment, the EDC-crosslinking beads are gelatin beads.

[0228] Cross-linked beads may possess specific characteristics advantageous for spreading, attachment, or encapsulation. For example, the beads may have a porous surface and / or be substantially empty. The presence of pores provides an increased cell attachment surface that allows a greater number of cells to attach compared to a non-porous or smooth surface. Additionally, the porous structure can support host tissue integration with the porous beads, which supports the formation of new tissue. The beads have a particle size distribution that can be fitted to a Weibull plot corresponding to a typical particle distribution pattern. In one embodiment, the cross-linked beads have an average diameter of about 120 μm, about 115 μm, about 110 μm, about 109 μm, about 108 μm, about 107 μm, about 106 μm, about 105 μm, about 104 μm, about 103 μm, about 102 μm, about 101 μm, about 100 μm, about 99 μm, about 98 μm, about 97 μm, about 96 μm, about 95 μm, about 94 μm, about 93 μm, about 92 μm, about 91 μm, or less than about 90 μm. The characteristics of the cross-linked beads vary depending on the casting process. For example, the process of using airflow to aerosolize a liquid gelatin solution and spray it into liquid nitrogen using a thin-layer chromatography reagent sprayer (ACE Glassware) is used to provide beads having the aforementioned characteristics. A person skilled in the art will recognize that adjusting the parameters of the casting process provides an opportunity to match different characteristics of the beads, such as different particle size distributions.

[0229] The cytocompatibility of cross-linked beads is evaluated in vitro prior to preparation using cell culture technology in which the beads are cultured with cells corresponding to the final bioactive cell preparation. For example, the beads are cultured with primary kidney cells prior to the preparation of a bioactive kidney cell preparation, and a living / dead cell assay is used to confirm cytocompatibility. In certain preparations, biocompatible cross-linked beads are combined with a temperature-sensitive biomaterial in solution at a solution volume of about 5% (w / w) to about 15% (w / w). The crosslinked beads may be present in a solution volume of about 5% (w / w), about 5.5% (w / w), about 6% (w / w), about 6.5% (w / w), about 7% (w / w), about 7.5% (w / w), about 8% (w / w), about 8.5% (w / w), about 9% (w / w), about 9.5% (w / w), about 10% (w / w), about 10.5% (w / w), about 11% (w / w), about 11.5% (w / w), about 12% (w / w), about 12.5% ​​(w / w), about 13% (w / w), about 13.5% (w / w), about 14% (w / w), about 14.5% (w / w), or about 15% (w / w).

[0230] In another aspect, the present disclosure provides a formulation containing a biomaterial that degrades over a period of approximately minutes, hours, or days. This contrasts with much of the work that focuses on the implantation of solid materials that degrade slowly over the following weeks or months.

[0231] In another aspect, the present disclosure provides a formulation having biocompatible cross-linked beads inoculated with bioactive cells together with a delivery matrix. In one embodiment, the delivery matrix has one or more of the following features: being biocompatible, biodegradable / bioabsorbable, substantially solid, having lost structural integrity (substantially solid) after implantation, and a cytocompatible environment to support cell viability prior to or during implantation into a target. The ability of the delivery matrix to maintain spaced implanted particles (e.g., cross-linked beads) during implantation promotes intrinsic tissue growth. In the absence of a delivery matrix, compression of the beads into small compartments during implantation may result in unsuitable space for sufficient tissue growth. The delivery matrix facilitates the implantation of solid formulations. Additionally, the short duration of structural integrity means that immediately after implantation, the matrix does not provide a significant barrier to the tissue growth or integration of the delivered cells / materials with the host tissue. The delivery matrix provides localization of the formulation described herein because the embedded solid units help prevent the delivered material from dispersing into the tissue during transplantation. For cell-based formulations, the solid delivery matrix improves the stability and viability of anchorage-dependent cells compared to cells suspended in fluid.

[0232] In one embodiment, the delivery matrix is ​​a population of biocompatible beads that are not smeared onto cells. In another embodiment, the unsmeared beads are dispersed between individual cells and smeared beads and throughout the beads. The unsmeared beads act as "spacer beads" between the cells and smeared beads prior to and immediately after transplantation. The spacer beads contain a temperature-sensitive biomaterial that is substantially solid at a first temperature and substantially liquid at a second temperature, wherein the first temperature is lower than the second temperature. For example, as described herein, the spacer beads contain a biomaterial that is substantially solid at about ambient temperature or below and substantially liquid at about 37°C. In one embodiment, the ambient temperature is about room temperature. In another embodiment, the biomaterial is a gelatin solution. The gelatin solution is present in an amount of about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, or about 11% (w / v). The gelatin solution may be provided in PBS, cell culture medium (e.g., DMEM), or another suitable solvent.

[0233] In one aspect, the present disclosure provides a formulation containing a biomaterial that is implanted in a substantially solid form (e.g., a spacer bead) and then liquefies / melts or otherwise loses structural integrity after implantation into the body. This contrasts with a significant portion of the work that focuses on the use of materials that can be injected as liquids, where said material coagulates within the body after injection.

[0234] The temperature sensitivity of spacer beads can be evaluated in vitro prior to preparation. Spacer beads can be labeled and mixed with unlabeled non-temperature-sensitive beads. The mixture is then incubated at 37°C to observe changes in physical transition. Loss of shape in labeled temperature-sensitive beads is observed over time at higher temperatures. For example, temperature-sensitive gelatin beads are made with Alcian blue dye and can serve as markers for physical transition. Blue gelatin beads are mixed with Cultispher S beads (white), loaded into a catheter, then extruded at 37°C in 1X PBS, pH 7.4, and incubated. Loss of shape in blue gelatin beads occurs microscopically at different time points. Changes in the physical state of the blue gelatin beads can be observed after 30 minutes, and this becomes more pronounced with extended incubation times. The beads do not completely disappear due to the viscosity of the material.

[0235] The bioactive cell preparations described herein may be used to manufacture renal cell-based preparations for injection into the kidney. However, a person skilled in the art will recognize that the preparations would be suitable for many other types of bioactive cell populations. For example, the present disclosure considers preparations for bioactive cells for injection into any solid organ or tissue.

[0236] In one aspect, the bioactive cell preparation described herein will contain a fixed number of cells. In one embodiment, the total number of cells for the preparation is about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , or about 10 9There are individual cells. In one embodiment, the dosage of cells for the formulation described herein may be calculated based on the estimated mass or functional mass of the target organ or tissue. In a specific embodiment, the bioactive cell formulation contains a dosage corresponding to the number of cells based on the weight of the host organ to be treated with the formulation. For example, the bioactive kidney cell formulation is based on an average weight of about 150 grams for a human kidney. . In one embodiment, the number of cells per gram (g) of the kidney is about 600 cells / g to about 7.0 x 10⁶ 7 The cell count is per gram. In some embodiments, the cell count per gram of the kidney is about 600 cells / g, about 1000 cells / g, about 1500 cells / g, about 2000 cells / g, about 2500 cells / g, about 3000 cells / g, about 3500 cells / g, about 4000 cells / g, about 4500 cells / g, about 5000 cells / g, about 5500 cells / g, about 6000 cells / g, about 6500 cells / g, about 7000 cells / g, about 7500 cells / g, about 8000 cells / g, about 8500 cells / g, about 9000 cells / g, about 9500 cells / g, or about 10,000 cells / g.

[0237] In another embodiment, the number of cells per gram of kidney is approximately 1.5 x 10⁶ 4 Dog cells / g, approx. 2.0 x 10⁶ 4 Dog cells / g, approx. 2.5 x 10⁶ 4 Dog cells / g, approx. 3.0 x 10⁶ 4 Dog cells / g, approx. 3.5 x 10⁶ 4 Dog cells / g, approx. 4.0 x 10⁶ 4 Dog cells / g, approx. 4.5 x 10⁶ 4 Dog cells / g, approx. 5.0 x 10⁶ 4 Dog cells / g, approx. 5.5 x 10⁶ 4 Dog cells / g, approx. 6.0 x 10⁶ 4 Dog cells / g, approx. 6.5 x 10⁶4 Dog cells / g, approx. 7.0 x 10⁶ 4 Dog cells / g, approx. 7.5 x 10 4 Dog cells / g, approx. 8.0 x 10⁶ 4 Dog cells / g, approx. 9.5 x 10 4 It is one cell / g.

[0238] In another embodiment, the number of cells per gram of kidney is approximately 1.0 x 10⁶ 5 Dog cells / g, approx. 1.5 x 10 5 Dog cells / g, approx. 2.0 x 10⁶ 5 Dog cells / g, approx. 2.5 x 10⁶ 5 Dog cells / g, approx. 3.0 x 10⁶ 5 Dog cells / g, approx. 3.5 x 10⁶ 5 Dog cells / g, approx. 4.0 x 10⁶ 5 Dog cells / g, approx. 4.5 x 10⁶ 5 Dog cells / g, approx. 5.0 x 10⁶ 5 Dog cells / g, approx. 5.5 x 10⁶ 5 Dog cells / g, approx. 6.0 x 10⁶ 5 Dog cells / g, approx. 6.5 x 10⁶ 5 Dog cells / g, approx. 7.0 x 10⁶ 5 Dog cells / g, approx. 7.5 x 10 5 Dog cells / g, approx. 8.0 x 10⁶ 5 Dog cells / g, approx. 8.5 x 10 5 Dog cells / g, approx. 9.0 x 10⁶ 5 Dog cells / g, or about 9.5 x 10⁶ 5 It is one cell / g.

[0239] In another embodiment, the number of cells per gram of kidney is approximately 1.0 x 10⁶ 6 Dog cells / g, approx. 1.5 x 10 6 Dog cells / g, approx. 2.0 x 10⁶ 6 Dog cells / g, approx. 2.5 x 10⁶ 6 Dog cells / g, approx. 3.0 x 10⁶ 6 Dog cells / g, approx. 3.5 x 10⁶ 6Dog cells / g, approx. 4.0 x 10⁶ 6 Dog cells / g, approx. 4.5 x 10⁶ 6 Dog cells / g, approx. 5.0 x 10⁶ 6 Dog cells / g, approx. 5.5 x 10⁶ 6 Dog cells / g, approx. 6.0 x 10⁶ 6 Dog cells / g, approx. 6.5 x 10⁶ 6 Dog cells / g, approx. 7.0 x 10⁶ 6 Dog cells / g, approx. 7.5 x 10 6 Dog cells / g, approx. 8.0 x 10⁶ 6 Dog cells / g, approx. 8.5 x 10 6 Dog cells / g, approx. 9.0 x 10⁶ 6 Dog cells / g, approx. 9.5 x 10 6 Dog cells / g, 1.0 x 10⁶ 7 Dog cells / g, or about 1.5 x 10⁶ 7 It is one cell / g.

[0240] The total number of cells can be selected for the formulation, and the volume of the formulation can be adjusted to reach an appropriate dosage.

[0241] In some embodiments, the formulation may contain a dose of cells for the subject, which is a single dose or a single dose plus an additional dose. In other embodiments, the dose may be provided by a structure as described herein. The therapeutically effective amount of a renal cell population or a mixture of renal cell populations as described herein may range from the maximum number of cells safely accommodated by the subject to the minimum number of cells essential for the treatment of renal disease, e.g., stabilization of one or more renal functions, a reduced rate of decline, or improvement.

[0242] A therapeutically effective amount of the renal cell population or mixture thereof described herein may be suspended in a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, basal culture medium plus 1% serum albumin, saline, buffered saline, dextrose, water, collagen, alginate, hyaluronic acid, fibrin glue, polyvinyl alcohol, carboxymethylcellulose, and combinations thereof. The formulation must be suitable for the mode of administration.

[0243] Accordingly, the present disclosure provides for the use of a formulation containing a kidney cell population or a mixture thereof, e.g., a B2 cell population alone or a mixture with a B3 and / or B4 or B4' cell population, for the preparation of a drug for treating kidney disease in a subject. In some embodiments, the drug further comprises a recombinant polypeptide, e.g., a growth factor, a chemokine, or a cytokine. In additional embodiments, the drug comprises a human kidney-derived cell population. The cells used to prepare the drug may be isolated, derived, or concentrated using any rate of change provided for the method provided herein.

[0244] The renal cell product(s), or mixtures or compositions thereof, are prepared according to routine procedures as pharmaceutical compositions adapted for administration to humans. Typically, compositions for intravenous administration, intra-arterial administration, or administration into renal capsules are, for example, solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a local anesthetic to alleviate any pain at the injection site. Generally, the components are supplied as unit formulations, for example, as concentrates cryopreserved in sealed containers, such as ampoules indicating the amount of active agent, either mixed together or separately. When the composition is to be administered by infusion, it may be dispensed into an infusion vial containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water or saline for injection may be provided so that the components can be mixed prior to administration.

[0245] Pharmaceutically acceptable carriers are determined in part by the specific composition being administered and by the specific method used to administer the composition. Accordingly, there is a wide variety of formulations suitable for pharmaceutical compositions (see, e.g., Alfonso R Gennaro (ed.), Remington: The Science and Practice of Pharmacy, formerly Remington's Pharmaceutical Sciences 20th ed., Lippincott, Williams & Wilkins, 2003, which is incorporated herein by reference in its entirety). Pharmaceutical compositions are generally prepared as sterile, substantially isotonic formulations and fully comply with all U.S. Food and Drug Administration Good Manufacturing Practice (GMP) regulations.

[0246] One aspect provides a pharmaceutical formulation comprising a kidney cell product, e.g., a B2 cell product alone or in combination with a B3 and / or B4 or B4' cell product, and a pharmaceutically acceptable carrier. In some embodiments, the formulation comprises 10 4 to 10 9 It contains canine mammalian kidney-derived cells.

[0247] Modified release agent

[0248] In one aspect, the formulation of the present disclosure is provided as a modified release formulation. Generally, the modified release is characterized by an initial release of a first activator upon administration, followed by at least one additional, subsequent release of a second activator. The first and second activators may be the same or they may be different. In one embodiment, the formulation provides modified release through multiple components in the same formulation. In another embodiment, the modified release formulation contains an activator as part of a first component that allows the activator to move freely throughout the volume of the formulation, thereby allowing for immediate release to a target site upon administration. The first component may be a temperature-sensitive biomaterial having substantially a liquid phase and substantially a solid phase, and when in the solid phase, the first component is substantially in the liquid phase at the time of administration. In one embodiment, the activator is substantially in the liquid phase so as to move substantially freely throughout the volume of the formulation and is therefore immediately released to a target site upon administration.

[0249] In another embodiment, the modified release formulation has an activator as a portion of a second component that is attached to, deposited on, coated with, embedded within, smeared on, or captured within the second component, which persists before and after administration to the target site. The second component contains a structural element capable of associating with the activator and thus prevents the immediate release of the activator from the second component at the time of administration. For example, the second component is provided in a substantially solid form, such as biocompatible beads, which can be cross-linked to prevent or delay enzymatic degradation in vivo. In one embodiment, the activator in a substantially solid form retains its structural integrity within the formulation before and after administration and thus does not immediately release the activator to the target site at the time of administration. Although a carrier suitable for the modified release formulation has been described herein, a person skilled in the art will recognize other carriers suitable for use in the present specification.

[0250] In one embodiment, the formulation provides rapid initial delivery / release of the delivered element, including cells, nanoparticles, therapeutic molecules, etc., and subsequent delayed release of the element. The formulation of the present disclosure may be designed for such a two-phase release profile in which the agent to be delivered is provided in both a non-attached form (e.g., cells in solution) and an attached form (e.g., cells attached to beads or another suitable carrier). At the initial administration, the non-interfering agent is immediately delivered to the delivery site, whereas the release of the interfering agent is delayed until the structural integrity of the carrier (e.g., beads) fails at the time when the previously attached agent is released. As discussed below, other suitable mechanisms of release will be recognized by those skilled in the art.

[0251] The time delay for release can be adjusted based on the properties of the activator. For example, in bioactive cell preparations, the time delay for release may be approximately a few seconds, minutes, hours, or days. In some environments, a delay of approximately a few weeks may be appropriate. For other activators, such as small molecules or macromolecules, the time delay for release in the preparation may be approximately a few seconds, minutes, hours, days, weeks, or months. It is also possible for the preparation to contain different biomaterials that provide different time-delayed release profiles. For example, a first activator and a first biomaterial may have a first release time, and a second activator and a second biomaterial may have a second release time. The first and second activators may be the same or different.

[0252] As discussed herein, the duration of delayed release may generally correspond to the duration of the loss of structural integrity of the biomaterial. However, a person skilled in the art will recognize other mechanisms of delayed release. For example, the activator may continue to be released over time regardless of the degradation of any specific biomaterial, such as the diffusion of the drug from the polymer matrix. Additionally, the bioactive cell may leave the preparation containing the biomaterial and the bioactive cell and migrate to the native tissue. In one embodiment, the bioactive cell migrates from the biomaterial, such as beads, to the native tissue.

[0253] Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Extended absorption of injectable formulations may be induced by including agents that delay absorption in the formulation, e.g., monostearate salts and gelatin. Many methods for preparing such formulations are patented or are generally known to those skilled in the art. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. Additional methods applicable to the controlled or extended release of polypeptide agents are described, for example, in U.S. Patent Nos. 6,306,406 and 6,346,274, and, for example, in U.S. Patent Application Nos. US20020182254 and US20020051808, all of which are incorporated herein by reference.

[0254] 9. Method and Route of Administration

[0255] The bioactive cell preparation of the present disclosure may be administered alone or in combination with other bioactive components. The preparation is suitable for the injection or implantation of tissue manipulation elements incorporated into the interior of solid organs to regenerate tissue. Additionally, the preparation is used for the injection or implantation of tissue manipulation elements into the walls of hollow organs to regenerate tissue.

[0256] In one aspect, the present disclosure provides a method for providing the bioactive cell preparation described herein to a subject in need thereof. In one embodiment, the source of the bioactive cells may be autologous, or allogeneic, homogeneous (autologous genotype or allogeneic genotype), and any combination thereof. Where the source is not autologous, the method may include the administration of an immunosuppressive agent. Suitable immunosuppressive drugs include azathioprine, cyclophosphamide, mizorvine, cyclosporine, tacrolimus hydrate, chlorambucil, lovezarit disodium, auranofin, alprostadil, gusperimus hydrochloride, biosynsorb, muromonav, alefacept, pentostatin, daclizumab, sirolimus, mycophenolate mofetil, leflonamide, basiliximab, dornase α, vindarid, cladribine, pimecrolimus, ilodecakin, cedelizumab, epalizumab, everolimus, anisperimus, gavilimomab, paralimomab, cloparabine, rapamycin, siplizumab, cyreito, LDP-03, CD4, SR-43551. SK&F-106615, IDEC-114, IDEC-131, FTY-720, TSK-204, LF-080299, A-86281, A-802715, GVH-313, HMR-1279, ZD-7349, IPL-423323, CBP-1011, MT-1345, CNI-1493, CBP-2011, J-695, LJP-920, L-732531, ABX-RB2, AP-1903, IDPS, BMS-205820, BMS-224818, CTLA4-1g, ER-49890, ER-38925, ISAtx-247, RDP-58, PNU-156804, LJP-1082, TMC-95A, TV-4710, PTR-262-MG, and AGI-1096 (see U.S. Patent No. 7,563,822) are included, but not limited to. A person skilled in the art will recognize other suitable immunosuppressive drugs.

[0257] The therapeutic method of the present disclosure comprises the delivery of the bioactive cell preparation described herein. In one embodiment, direct administration of the cells to the site of intended benefit is preferred. A subject requiring this may be treated by bringing the native kidney into in vivo contact with the bioactive cell preparation described herein, together with a product secreted from one or more concentrated kidney cell populations and / or a mixture or structure comprising the same.

[0258] The step of bringing the intrinsic kidney into in vivo contact with the secreted product can be achieved through the use / administration of a preparation containing a population of the secreted product from a cell culture medium, e.g., a conditioning medium, or by the transplantation of a concentrated cell population and mixture or structure capable of secreting the product in vivo. The in vivo contact step provides a regenerative effect on the intrinsic kidney.

[0259] Various means for administering cells and / or secreted products to a subject will be apparent to a person skilled in the art in consideration of this description. Such methods include injecting cells into the target of the subject.

[0260] Cells and / or secreted products may be inserted into a delivery device or vehicle that facilitates introduction into a subject by injection or implantation. In certain embodiments, the delivery vehicle may contain natural materials. In certain embodiments, the delivery vehicle may contain synthetic materials. In one embodiment, the delivery device provides a structure similar to or appropriately fitting to the structure of an organ. In another embodiment, the delivery vehicle is virtually similar to a fluid. Such a delivery device may include a tube, such as a catheter, for injecting cells and fluids into the body of a recipient. In a preferred embodiment, the tube additionally has a needle, such as a syringe, through which cells may be introduced to a desired location in the subject. In some embodiments, a mammalian kidney-derived cell population is prepared for administration into blood vessels through a catheter (the term "catheter" is intended to include any various tubular systems for delivering substances into blood vessels). Alternatively, cells may be embedded in or on biomaterials or scaffolds, including but not limited to fabrics, such as weaves, knits, braids, meshes, and non-woven fabrics, perforated films, sponges, foams, and beads, such as solid or porous beads, microparticles, nanoparticles, etc. (e.g., Cultispher-S gelatin beads - Sigma). Cells may be prepared for delivery in various different forms. For example, cells may be suspended in a solution or gel. Cells may be mixed with pharmaceutically acceptable carriers or diluents, while the cells remain alive. Pharmaceutically acceptable carriers and diluents include saline solution, aqueous buffer solution, solvent, and / or dispersion medium. The uses of such carriers and diluents are well known in the art. The solution will preferably be sterile, fluid, and often isotonic.Preferably, the solution is stable under conditions of preparation and storage and is preserved from contamination by microorganisms such as bacteria and fungi through the use of, for example, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. A person skilled in the art will recognize that a delivery vehicle used in the delivery of a cell population and a mixture thereof may include a combination of the features mentioned above.

[0261] The methods of administration of a preparation containing isolated renal cell population(s), e.g., a B2 cell population alone or a cell population mixed with B4' and / or B3, include, but are not limited to, systemic, intrarenal (e.g., parenchyma), intravenous or intra-arterial injection, and direct injection into tissue at the intended active site. Additional methods of administration to be used include single or multiple injection(s) via direct laparotomy, direct laparoscopy, transperitoneum, or percutaneous delivery. Other additional methods of administration to be used include, e.g., retrograde and ureteropelvic infusion. Surgical delivery means include, but are not limited to, 1-stage procedures including partial nephrectomy and structure transplantation, partial nephrectomy, partial pyelectomy, vascularization between the serosa and the peritoneum, multifocal biopsy needle track, cone or pyramidal to cylinder and renal rod-shaped replacement, as well as 2-stage procedures including, for example, a tracheo-like-intra-bioreactor for re-transplantation. In one embodiment, a preparation containing a cell mixture is delivered at the same time through the same route. In another embodiment, each cell composition containing a controlled mixture is delivered separately to a specific location or simultaneously or in a time-controlled manner through a specific method by one or more of the methods described herein.

[0262] The appropriate cell transplant dosage in humans can be determined from existing information regarding cell activity, e.g., EPO production, or extrapolated from dosage studies conducted in preclinical research. From in vitro cultures and in vivo animal experiments, the amount of cells is quantified and can be used to calculate the appropriate dosage of the transplant material. Additionally, the patient may be monitored to determine whether additional transplants can be performed or if the transplant material should be reduced accordingly.

[0263] Selected extracellular matrix components, such as one or more types of collagen or hyaluronic acid known in the art, and / or one or more other components, including growth factors, platelet-rich plasma, and drugs, may be added to the cell population of the present invention and mixtures thereof.

[0264] A person skilled in the art will recognize various formulations and methods of administration suitable for the secreted product described in this specification.

[0265] 10. Kits and Manufactured Products

[0266] The present disclosure further comprises a kit comprising a matrix and scaffold and related materials as disclosed herein, and / or a cell culture medium and instructions for use. The instructions for use may include, for example, instructions for cell culture or instructions for administering cells and / or cell products. In one embodiment, the present disclosure provides a kit comprising a scaffold and instructions as described herein. In another embodiment, the kit comprises an agent for detecting marker expression, a reagent for using the agent, and instructions for use. The kit may be used for the purpose of determining the prognosis of regeneration of the intrinsic kidney in a subject after transplantation or administration of the cell population, mixture, or structure described herein. The kit may also be used to determine the biotherapeutic efficacy of the cell population, mixture, or structure described herein.

[0267] Another embodiment is a manufactured product containing bioactive cells useful for treating a subject requiring treatment. The manufactured product comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, glass vials, syringes, etc. The container may be formed from various materials such as glass or plastic. The container may contain a composition effective for treating a disease and may have a sterile access port (for example, the container may be a fluid bag or glass vial with a stopper that can be punctured by a needle). At least one active agent in the formulation is a population of bioactive cells as provided herein. The label or package insert specifies that the formulation is used to treat a specific disease. The label or package insert will further include instructions for administering the formulation to a patient. Manufactured products and kits including combination therapies described herein are also considered. The drug information refers to the customary information included in the commercial package of the therapeutic product containing information regarding indications, dosage, administration, contraindications, and / or warnings concerning the use of such therapeutic product. In one embodiment, the drug information specifies that the preparation is used to treat a disease or disorder, such as, for example, kidney disease or disorder. It may additionally include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes. For various purposes, kits useful for, for example, for evaluating regenerative results are also provided. As described herein, kits containing detection agents for urine-derived vesicles and / or their contents, such as nucleic acids (e.g. miRNA), vesicles, exosomes, etc., may be provided.Detection agents include, but are not limited to, nucleic acid primers and probes, and antibodies for in vitro detection of a desired target. As with manufactured products, the kit includes a container and a label or pharmaceutical information on or associated with the container. The container holds a composition containing at least one detection agent. For example, additional containers containing a diluent and buffer or a control detection agent may be included. The label or pharmaceutical information will provide a description of the composition and instructions for the intended in vitro prognosis or diagnostic use.

[0268] 11. Report

[0269] When practiced for commercial purposes, the method of the present disclosure generally provides a report or summary regarding the prognosis of regeneration. The method of the present disclosure will provide a report containing a prediction of the development or outcome of regeneration before and after any administration or transplantation of a preparation containing the cell population, mixture, or structure described herein. The report may include information on any indicators related to the prognosis. The method and report of the present disclosure may further include storing the report in a database. Alternatively, the method may further create a record in a database regarding the subject and append the record along with the data. In one embodiment, the report is a paper report; in another embodiment, the report is an auditory report; and in yet another embodiment, the report is an electronic record. The report is considered to be provided to a physician and / or patient. Receiving the report may further include establishing a network connection to a server computer containing the data and the report and requesting the data and the report from the server computer. Additionally, the method provided herein may be automated in whole or in part.

[0270] The invention as exemplarily described herein may be practiced in the absence of any elements or elements, limitations or restrictions not specifically disclosed herein. Accordingly, for example, in each example of this specification, any one of the terms “comprising,” “essentially composed,” and “composed” may be replaced with either of the other two terms. Accordingly, for an embodiment of the invention using one of the terms, the invention may also include another embodiment in which one of these terms may be replaced with another of these terms. In each embodiment, the terms have their established meanings. Accordingly, for example, one embodiment may include a formulation “comprising” a plurality of components, another embodiment may include a formulation “essentially composed” of the same components, and a third embodiment will include a formulation “composed” of the same components. It is acknowledged that the terms and expressions used are for descriptive rather than limiting purposes, and are not intended to exclude any equivalents or parts thereof of the features shown and described in the use of such terms and expressions, but that various modifications are possible within the scope of the claimed invention. Accordingly, it should be understood that, although the invention has been specifically disclosed by preferred embodiments and optional features, variations and modifications of the concepts disclosed herein may be relied upon by those skilled in the art, and that such variations and modifications are considered within the scope of the invention as defined by the appended claims.

[0271] The foregoing description is deemed sufficient for those skilled in the art to practice the present invention. The following examples are provided solely for illustrative purposes and are not intended to limit the scope of the invention in any way. In fact, in addition to those shown and described herein, various variations of the invention will become apparent to those skilled in the art from the foregoing description and will be included within the scope of the appended claims.

[0272] All patents, patent applications, and references cited in this specification are incorporated herein by reference in their entirety.

[0273] Examples

[0274] Example 1

[0275] Preparation of solution

[0276] This embodiment provides the composition of various culture medium formulations and solutions used in the following examples for the isolation and characterization of heterogeneous kidney cell populations and for the preparation of regenerative therapy products.

[0277] <Table 1.1>

[0278]

[0279] Dulbecco phosphate-buffered saline (DPBS) was used for washing all cells.

[0280] Example 2

[0281] Isolation of a heterogeneous non-dividing elongation cell population

[0282] This embodiment describes the isolation of an undivided (UNFX) heterogeneous kidney cell population from humans. A heterogeneous cell suspension was prepared from human kidney tissue by performing initial tissue dissociation.

[0283] Kidney tissue obtained through kidney biopsy provided source material for a heterogeneous renal cell population. Kidney tissue containing one or more of cortical, corticomedullary junction, or medullary tissue may be used. It is preferable to use corticomedullary junction tissue. Multiple biopsy cores (at least 2), avoiding scar tissue, were required from CKD kidneys. Clinical investigators obtained kidney tissue from patients at the hospital approximately 4 weeks prior to the planned transplantation of the final NKA. The tissue was transferred to the tissue transport medium of Example 1.

[0284] Afterwards, before processing the tissue for cell extraction, the tissue was washed with the tissue washing solution of Example 1 to reduce the biological burden.

[0285] Kidney tissue was chopped, weighed, and dissociated in the dissociation solution of Example 1. The resulting cell suspension was neutralized in Dulbecco Modified Eagle Medium (D-MEM) + 10% Fetal Bovine Serum (FBS) (Invitrogen, Carlsbad, CA), washed, and resuspended in serum-free, supplement-free Keratinocyte Medium (KSFM) (Invitrogen). Subsequently, a 15% (w / v) iodisanol (OptiPrep™, Sigma) gradient was prepared in the cell suspension and cm in the kidney cell growth medium of Example 1. 2 Red blood cells and debris were removed prior to the start of culture on tissue-treated polystyrene flasks or dishes at a density of 25,000 cells per cell. For example, 25 x 10 cells in 150 ml of 50:50 medium 6 Can be spread onto a T500 Nunc flask using individual cells / flasks.

[0286] Example 3

[0287] Cells of the isolated kidney cell population

[0288] Kidney cell expansion is based on the volume of tissue received and the success of isolating kidney cells from the resulting tissue. If necessary, the isolated cells can be cryopreserved (see below). Kidney cell growth kinetics may vary from sample to sample due to intrinsic variables of the cells isolated from individual patients.

[0289] The defined cell expansion process Table 3.1 It was developed to adjust the range of cell recovery due to the variability of the formed tissue. The expansion of kidney cells is achieved using defined cell culture procedures. Table 1.1 Closed culture vessels (e.g., T-flasks, Cell Factories, HyperStacks) in elongation cell growth medium ® Includes continuous series in ).

[0290] For human clinical trials, a BPE-free medium was developed to eliminate the inherent risks associated with the use of BPE. Cell growth, phenotype (CK18), and cellular function (GGT and LAP enzyme activity) were evaluated in the BPE-free medium and compared to the BPE-containing medium used in animal studies. Elongated cell growth, phenotype, and function were identical in both media. (Data not shown).

[0291] <Table 3.1>

[0292]

[0293] Once cell growth was observed in the initial T-flask (passage 0) and visual signs of contamination disappeared, the culture medium was replaced, and thereafter it was changed every 2-4 days. Fig. 2BThe elongation cell morphology was confirmed by evaluating the cells through visible observation of the culture under a microscope. The culture characteristically demonstrated a dense pavement or gravel shape due to the clumping of cells. These morphological features change during expansion and may not be present at every passage. Cell culture affineness was evaluated using an image library of cells at various levels of affineness in the culture baths used throughout the entire cell expansion.

[0294] When the culture vessel was at least 50% confluence, the elongated cells were subcultured by trypsinization. Fig. 2B . The detached cells were collected into tubes containing elongated cell growth medium, counted, and cell viability was calculated. In each cell passage, cells were plated at 500–4000 cells / cm2 in a sufficient number of culture vessels to expand the cell number to the level required for the NKA formulation. Fig. 2B The culture vessel was placed in a 37°C incubator in a 5% CO2 environment. As described above, cell morphology and fusion were monitored, and the tissue culture medium was replaced every 2-4 days. Table 3.2 This lists the viability of human kidney cells observed during cell isolation and expansion of six kidney biopsies from human donors.

[0295] <Table 3.2>

[0296]

[0297] Intrinsic variables of tissues from different patients resulted in different cell yields in culture. Therefore, it is not practical to strictly define the timing of cell passages or the number and type of culture vessels required at each passage to obtain the target number of cells. Typically, elongated cells undergo two or three passages; however, the culture duration and cell yield may vary depending on the cell growth rate. This is illustrated by the culture duration and cell yields (calculated) from six patients. Fig. 3It is exemplified in.

[0298] Cells were detached with EDTA and 0.25% trypsin (Invitrogen) for harvesting or passage. Viability was assessed by trypan blue exclusion, and hemacytometers or automated cellometers were used. ® Enumeration was performed manually using a counting system (Nexcelom Bioscience, Lawrence MA).

[0299] Example 4

[0300] Cryopreservation of cultured cells

[0301] Extended kidney cells were transiently cryopreserved to match the intrinsic variables of cell growth from individual patients and deliver the product according to a scheduled clinical schedule ( Fig. 2B Cryopreserved cells also provide an alternative source of cells when another NKA is required (e.g., patient disease, delays due to unexpected progression). Conditions used to cryopreserve cells and recover viable, functional cells upon thawing were established.

[0302] For cryopreservation, cells in a cryopreservation solution of approximately 50 x 10 6 Suspend to a final concentration of cells / mL ( Example 1 Note) And distributed into glass jars. Approximately 50x10 6 A 1 mL glass vial containing cells / mL was placed in the freezing chamber of a controlled-rate freezer and frozen at a pre-programmed rate. After freezing, the cells were transferred to a liquid nitrogen freezer for in-process storage.

[0303] Example 5

[0304] Preparation of SRC cell population

[0305] Fig. 2BDepending on the schedule management, selected kidney cells (SRC) can be produced from the final culture vessel grown from cryopreserved cells or directly from the extended culture.

[0306] If cryopreserved cells are used, the cells are thawed and plated onto tissue culture vessels for a final expansion step. When the final culture vessel is approximately 50–100% confluence, the cells are ready for processing for SRC isolation. Medium exchange and final washing with NKA dilute any residual cryopreservation solution in the final product.

[0307] Once the final cell culture vessel reached at least 50% confluence, the vessel was transferred to a hypoxic incubator set to 2% oxygen in a 5% CO2 environment at 37°C and cultured overnight. Fig. 2C Refer to [link]. Cells were maintained in an oxygen-controlled incubator set to 2% oxygen for as long as 48 hours. Exposure to a more physiologically relevant hypoxic (2%) environment improved cell separation efficacy and enabled greater detection of hypoxia-induced markers, such as VEGF.

[0308] After exposing cells to oxygen under hypoxic conditions for a sufficient period (e.g., overnight to 48 hours), cells were detached using EDTA and 0.25% trypsin (Invitrogen). Viability was assessed by trypan blue exclusion, and then using a hemacytometer or an automated cellometer. ® Counting was performed manually using a counting system (Nexcelom Bioscience, Lawrence, MA). Cells were washed once with DPBS, and approximately 850 x 10⁶ cells were placed in DPBS. 6 Resuspended down to the cell / mL level.

[0309] Density gradient centrifugation was used to separate the harvested kidney cell population based on cell buoyancy density. The kidney cell suspension was centrifuged in a single-step 7% iodisanol density gradient solution (OptiPrep in OptiMEM; 60% (w / v); Example 1 Separated from the (refer to)

[0310] A 7% OptiPrep gradient solution was prepared, and the refractive index indicating the desired density was measured prior to use (RI 1.3456 + / - 0.0004). Harvested kidney cells were layered on top of the gradient solution. The density gradient was centrifuged at room temperature for 20 minutes in either a centrifuge tube or a cell processor (e.g., COBE 2991) at 800 g It was centrifuged. Cell fractions exhibiting a buoyancy density greater than approximately 1.045 g / mL were collected as distinct pellets after centrifugation. Fig. 4 Cells maintaining a buoyancy density of less than 1.045 g / mL were excluded and discarded.

[0311] SRC pellets were resuspended in DPBS ( Fig. 2C Residual OptiPrep, FBS, culture medium, and incidental substances in the final product are minimized with four DPBS washes and one gelatin solution step.

[0312] Example 6

[0313] Cells with therapeutic potential can be isolated and proliferated from normal and chronically diseased kidney tissue.

[0314] The purpose of this study was to determine the functional characterization of human NKA cells through high-content analysis (HCA). High-content imaging (HCI) provides simultaneous imaging of multiple subcellular events using two or more fluorescent probes (multiprocessing) across multiple samples. High-content analysis (HCA) provides simultaneous quantitative measurement of multiple cellular parameters captured in high-content images. Briefly, undivided (UNFX) cultures were prepared from core biopsies taken from five human kidneys with late-stage chronic kidney disease (CKD) and three non-CKD human kidneys using standard biopsy procedures (Aboushwareb et al ., World J Urol 26, 295, 2008) and maintained it independently. After (2) passages of UNFX in vitro, cells were harvested (Basu et al A density gradient method (as described in Example 2 of WO 2012 / 064369) was performed to produce subfractions including subfractions B2, B3, and / or B4.

[0315] Ilagan et al Human kidney tissue was obtained from non-CKD and CKD human donors as summarized in Table 10.1 of PCT / US2011 / 036347. Ilagan et al Figure 4 of PCT / US2011 / 036347 shows the histopathological features of HK17 and HK19 samples. In vitro cultures were established from all non-CKD (3 / 3) and CKD (5 / 5) kidneys. High-content analysis of albumin transport in human NKA cells defining the region of interest (ROI) was performed by Ilagan et al . This is shown in Figure 5 of PCT / US2011 / 036347 (HCA of albumin transport in human NKA cells). A quantitative comparison of albumin transport in NKA cells derived from non-CKD and CKD kidneys is presented in Ilagan et al. As shown in Fig. 6 of PCT / US2011 / 036347. Ilagan et al As shown in Figure 6 of PCT / US2011 / 036347, albumin transport is not attenuated in CKD-derived NKA cultures. A comparative analysis of marker expression between tubule-concentrated B2 and tubule-cell-depleted B4 subfractions was Ilagan et al . As shown in Fig. 7 (CK8 / 18 / 19) of PCT / US2011 / 036347.

[0316] Comparative functional analysis of albumin transport between tubule-concentrated B2 and tubule-depleted B4 subfractions is Ilagan et al This is shown in Figure 8 of PCT / US2011 / 036347. Subfraction B2 is concentrated in proximal tubular cells and thus exhibits increased albumin-transport function.

[0317] Albumin absorptionThe culture medium of cells grown to confluence in 24-well Collagen IV plates (BD Biocoat™) was replaced with phenol-free, red, serum-free, low-glucose DMEM (pr- / s- / lg DMEM) containing 1X antifungal / antibiotic and 2 mM glutamine for 18–24 hours. Immediately before the assay, cells were washed and incubated for 30 minutes with pr- / s- / lg DMEM + 10 mM HEPES, 2 mM glutamine, 1.8 mM CaCl2, and 1 mM MgCl2. Cells were exposed to 25 μg / mL rhodamine-conjugated bovine albumin (Invitrogen) for 30 minutes, washed with ice-cold PBS to stop endocytosis, and immediately fixed in 2% paraformaldehyde containing 25 μg / mL Hoechst nuclear dye. For inhibition experiments, 1 μM receptor-associated protein (RAP) (Ray Biotech, Inc., Norcross GA) was added 10 minutes prior to albumin addition. Microscopic imaging and analysis were performed using a BD Pathway™ 855 high-concentration bioimaging camera (Becton Dickinson) (Kelley et al . Am J Physiol Renal Physiol. 2010 Nov;299(5):F1026-39. See Epub Sep 8, 2010).

[0318] In conclusion, HCA provides cellular-level data and can elucidate population kinetics, namely gene or protein expression, that cannot be detected by other assays. Human renal tubular cells can be characterized as components of the human NKA prototype by utilizing a quantifiable in vitro HCA assay to measure albumin transport (HCA-AT) function. HCA-AT enabled comparative evaluation of cellular function, demonstrating that albumin transport-qualified cells were retained in NKA cultures derived from human CKD kidneys. Furthermore, it was shown that specific subfractions of the NKA culture, B2 and B4, differ in phenotype and function, with B2 corresponding to a tubular cell-enriched fraction possessing enhanced albumin transport activity. The B2 cell subpopulation from human CKD is phenotype- and functionally similar to rodent B2 cells that exhibited efficacy in vivo (as shown above).

[0319] Example 7

[0320] Pre-gradient hypoxic culture affects band distribution, composition, and gene expression

[0321] To determine the effect of oxygen conditions on the composition and distribution of prototypes B2 and B4, neo-elongation cell preparations from different species were exposed to different oxygen conditions prior to the gradient step. Kelley et al ., 2010, supraAs described in [reference], rodent neo-kidney enlargement (NKA) cell products (RK069) were established using standard procedures for rat cell isolation and culture initiation. All flasks were cultured at 21% (atmosphere) oxygen for 2–3 days. The medium was changed, and subsequently, half of the flasks were relocated to an oxygen-controlled incubator set to 2% oxygen, while the remaining flasks were kept under 21% oxygen conditions for an additional 24 hours. Cells were then harvested from each set of conditions using the standard enzymatic harvesting procedure described above. A gradient step was prepared according to standard procedures, and "standard oxygen" (21% oxygen) and "hypoxic" (2% oxygen) cultures were harvested separately and applied side-by-side to the same gradient step. Four bands and pellets were formed in both conditions, but the distribution of cells across the gradient differed between the 21% and 2% oxygen culture batches. (Table 7.1) Specifically, the yield of B2 increased under hypoxia, accompanied by a decrease in B3. Additionally, the expression of B4-specific genes (e.g., erythropoietin) was enhanced in the resulting gradient from hypoxia-cultured cells (Presnell et al . Fig. 73 of WO / 2010 / 056328).

[0322] Basu et al. As described in WO 2012 / 064369, canine NKA cell products (DK008) were established using standard procedures for canine cell isolation and culture (similar to rodent isolation and culture procedures). All flasks were cultured for 4 days under 21% (atmospheric) oxygen conditions, after which a subset of the flasks was transferred to hypoxia (2%) for 24 hours, while the remaining subset of flasks was maintained at 21%. Subsequently, each set of flasks was harvested, and the same gradient steps were performed. Similar to the rat results, hypoxia-cultured canine cells were distributed differently across the entire gradient compared to atmospheric oxygen-cultured canine cells. (Table 7.1) In other words, along with a decrease in distribution within B3, the yield of B2 increased with hypoxic exposure prior to the gradient.

[0323] <Table 7.1>

[0324]

[0325] The above data shows that pre-gradient exposure to hypoxia enhances the distribution of specialized cells specific to B4 as well as the composition of B2. Therefore, hypoxic culture, then the above Basu et al. Density-gradient separation as described in is an effective way to generate 'B2' and 'B4' cell populations across species.

[0326] Example 8

[0327] Characterization of a non-dividing mixture of kidney cells isolated from samples of patients with autoimmune glomerulonephritis

[0328] As described above, a non-dividing mixture of renal cells was isolated from patient samples of autoimmune glomerulonephritis. To determine the random genotypic composition of a specific subpopulation of enlarged renal cells isolated from renal tissue, quantitative real-time PCR (qRTPCR) analysis (Brunskill) was performed. et al Differential cell-type-specific and pathway-specific gene expression patterns in cell subfractions were identified using Ilagan. et al As shown in Table 6.1 of PCT / US2011 / 036347, HK20 is a patient sample of autoimmune glomerulonephritis. Ilagan et al As shown in Table 6.2 of PCT / US2011 / 036347, cells generated from HK20 are deficient in glomerular cells, as determined by qRTPCR.

[0329] Example 9

[0330] Genetic profiling of therapeutically relevant renal bioactive cell populations isolated from patients with focal segmental glomerulosclerosis

[0331] To determine the random genotypic composition of a specific subpopulation of enlarged renal cells isolated from renal tissue, quantitative real-time PCR (qRTPCR) analysis (Brunskill et al ., supra Differential cell-type-specific and pathway-specific gene expression patterns in cell subfractions were identified using (2008). The presence of glomerular cells in the B4 fraction at harvest was evaluated for human product HK023, derived from patients with focal segmental glomerulosclerosis (FSGS) in which most glomeruli were destroyed. To put it simply, undivided (UNFX) cultures were prepared (Aboushwareb et al ., World J Urol 26, 295, 2008) and were independently maintained from each (4) core biopsy taken from the kidney using a standard biopsy procedure. After (2) passages of UNFX in vitro, cells were harvested and Basu et al Subfractions, including subfraction B4, known to be enriched with endocrine, vascular, and glomerular cells based on work performed on rodent, dog, and other human samples, were produced by performing a density gradient method according to Example 6 of WO2012 / 064369.

[0332] Fractions B4 were harvested separately from each independent UNFX sample of HK023, appearing as distinct bands of cells with a buoyancy density of 1.063–1.091 g / mL. RNA was isolated from each sample and tested for the expression of podocin (glomerular cell marker) and PECAM (endothelial cell marker) by quantitative real-time PCR. As expected from biopsy-derived samples from patients with severe FSGS, the presence of podocin(+) glomerular cells in fractions B4 was inconsistent, with podocin being undetectable in two-quarters of the samples. In contrast, PECAM+ angiocytes were consistently present in fractions B4 of four-quarters of the biopsy-derived cultures. Thus, fractions B4 can be isolated from human kidneys in a severely diseased state at a density range of 1.063–1.091 g / mL.

[0333] <Table 9.1>

[0334]

[0335] Additionally, Ilagan et al As shown in Table 7.2 of PCT / US2011 / 036347, the human sample (HK018) showed non-detectable glucosine (glomerular marker) by qRTPCR after density gradient centrifugation.

[0336] Example 10

[0337] Enrichment / depletion of viable kidney cell types using fluorescence-activated cell sorting (FACS)

[0338] One or more isolated kidney cells can be concentrated using fluorescence-activated cell sorting (FACS), and / or one or more specific kidney cell types can be depleted from the isolated primary kidney tissue.

[0339] reagent: 70% ethanol; washing buffer (PBS); 50:50 renal cell medium (50% DMEM high glucose): 50% keratinocyte-SFM; 0.4% trypan blue; primary antibodies against target renal cell populations, e.g., CD31 against renal endothelial cells and nephrine against renal glomerular cells; matched isotype-specific fluorescent secondary antibodies; staining buffer (0.05% BSA in PBS).

[0340] procedure: After standard procedures for washing the biological safety cabinet (BSC), a single-cell suspension of kidney cells from the primary separation or cultured cells may be obtained from a T500 T / C-treated flask, resuspended in kidney cell medium, and placed on ice. Subsequently, cell number and viability are determined using the trypan blue exclusion method. For example, for the enrichment / depletion of kidney cells from glomerular or endothelial cells of a heterogeneous population, 10 to 50 x 10⁶ cells having at least 70% viability 6 Canine live cells are obtained. Subsequently, a heterogeneous population of kidney cells is treated with staining buffer at an initiation concentration of 1 μg / 0.1 ml / 1 x 10⁶ 6 Stain with a primary antibody specific to the target cell type in canine cells (at appropriate concentrations if necessary). The target antibody may be conjugated, for example, to CD31 PE (specific to renal endothelial cells) or not conjugated, for example, to nephrine (specific to renal glomerular cells).

[0341] Subsequently, the cells are stained for 30 minutes on ice or at 4°C protected from light. After 30 minutes of incubation, the cells were centrifuged at 300xg for 5 minutes. The pellet was then resuspended in PBS or staining buffer, depending on whether a conjugated isotype-specific secondary antibody is required. Once the cells are labeled with the fluorescent dye-conjugated primary antibody, the cells 10 7Resuspend each cell in 2 ml of PBS and perform sorting on a FACS aria or equivalent cell sorter. If the cells are not labeled with the fluorescent dye-conjugated antibody, use 1 µg / 0.1 ml / 10 6 Label with an isotype-specific conjugated secondary antibody at the initiation concentration of the cells.

[0342] Subsequently, the cells were stained for 30 minutes on ice or at 4°C protected from light. After 30 minutes of incubation, the cells were centrifuged at 300xg for 5 minutes. After centrifugation, the pellet was collected in 5x10 6 Resuspend in PBS at a concentration of / ml PBS, and then transfer 4ml per 12x75mm tube to a sterile tube.

[0343] The FACS Aria is prepared for sterile sorting of viable cells according to the manufacturer's instructions (BD FACS Aria User Manual). Load sample tubes onto the FACS Aria and adjust the PMT voltage after starting collection. Draw a gate to screen kidney-specific cell types using fluorescence intensity with a specific wavelength. Draw another gate to screen the negative population. Once the desired gates are drawn to encapsulate the positive and negative populations, sort the cells using the manufacturer's instructions.

[0344] The positive target population was collected in one 15 ml conical tube, and the negative population was collected in another 15 ml conical tube filled with 1 ml of renal cell medium. After collection, samples from each tube were analyzed by flow cytometry to determine purity. The collected cells were washed by centrifugation at 300 x g for 5 minutes, and the pellet was resuspended in renal cell medium for further analysis and experimentation.

[0345] Example 11

[0346] Concentration / Depletion of Kidney Cell Types Using Magnetic Cell Sorting

[0347] One or more isolated kidney cells can be concentrated and / or one or more specific kidney cell types can be depleted from the isolated primary kidney tissue.

[0348] reagent: 70% ethanol, washing buffer (PBS), 50:50 renal cell medium (50% DMEM high glucose): 50% keratinocyte-SFM, trypan blue 0.4%, running buffer (PBS, 2mM EDTA, 0.5% BSA), washing buffer (PBS, 2mM EDTA), washing solution (70% v / v ethanol), Miltenyi FCR blocking reagent, Miltenyi micro beads specific to IgG isotypes, target antibody, e.g., CD31 (PECAM) or nephrine, or secondary antibody.

[0349] procedure: After standard procedures for washing the biological safety cabinet (BSC), obtain a single-cell suspension of renal cells from the primary isolation or culture and resuspend them in renal cell medium. Determine cell number and viability using the trypan blue exclusion method. For example, for the enrichment / depletion of renal cells from glomerular or endothelial cells of a heterogeneous population, at least 10 cells with at least 70% viability 6 Up to 4x10 9 Obtain live cells up to the dog.

[0350] Determine the optimal separation for the enrichment / depletion approach based on the target cells of interest. For enrichment of glomerular cells with a target frequency of less than 10% using nephrine antibodies, use Miltenyi autoMACS or an equivalent device program POSSELDS (double-positive screening in sensitive mode). For depletion with a target frequency greater than 10%, use Miltenyi autoMACS or an equivalent device program DEPLETES (depletion in sensitive mode).

[0351] 1 μg / 10 live cells with 0.05% BSA in a 15 ml conical centrifuge tube 6 By adding 0.1 ml of PBS per cell, the cells are labeled with a target-specific primary antibody, e.g., nephrine rb polyclonal antibody, and then incubated at 4°C for 15 minutes.

[0352] After labeling, 10 7 1-2 ml of buffer was added per cell, and the cells were washed by centrifugation at 300xg for 5 minutes to remove unbound primary antibodies. After washing, 1 µg / 10 with 0.05% BSA 6 Add 0.1 ml of PBS isotype-specific secondary antibody, e.g. chicken anti-rabbit PE, and then incubate at 4°C for 15 minutes.

[0353] After incubation, 10 7 1-2 ml of buffer was added per cell, and the cells were washed by centrifuging at 300xg for the next 5 minutes to remove unbound secondary antibodies. The supernatant was removed, and the cell pellet was 10 7 Resuspend in 60 μl of buffer per total cell, and then 10 7 Add 20 μl of FCR blocking reagent per total cell and mix well.

[0354] Add 20 μl of direct MACS micro beads (e.g., anti-PE micro beads) and mix, then incubate at 4°C for 15 minutes.

[0355] After incubation, add 10-20x labeling volume of buffer and wash the cells by centrifuging the cell suspension at 300xg for 5 minutes, and 10 8 The cell pellet was resuspended in 500 μl–2 ml of buffer per cell.

[0356] In accordance with the manufacturer's instructions, clean the autoMACS system and prepare for self-cell separation using autoMACS. Place a new sterile collection tube under the outlet port. Select the autoMACS cell separation program. Select the POSSELDS program for screening. Select the DEPLETES program for depletion.

[0357] Insert the labeled cells into the uptake port and start the program. After cell screening or depletion, collect the samples and place them on ice until use. Verify the purity of the depleted or screened samples using flow cytometry.

[0358] Example 12

[0359] Phenotypic characterization of a concentrated heterogeneous kidney cell population

[0360] The following examples are Example 5 The use of flow cytometry to characterize selected heterogeneous human kidney cells is described in detail. The heterogeneous kidney cell population consists mainly of renal endothelial cells, which are well known for their regenerative capacity. Other parenchymal (vascular) and stromal (collecting duct) cells may be rarely present in the autologous cell population.

[0361] Cell phenotypes are monitored by analyzing the expression of kidney cell markers using flow cytometry. The analysis of cell phenotypes is based on the use of antigen markers specific to the cell type being analyzed. Flow cytometry provides a quantitative measurement of cells within a sample population expressing the antigen marker being analyzed.

[0362] Several markers useful for characterizing the phenotype of kidney cells have been reported in this literature: (i) cytokeratin; (ii) transport membrane proteins (aquaporins and curbilins); (iii) cell binding molecules (adherins, differentiation clusters, and lectins); and (iv) metabolic enzymes (glutathione). Since the majority of cells found in cultures derived from whole kidney degradation are epithelial and endothelial, the markers examined focus on the expression of proteins specific to these two groups.

[0363] Cytokeratin is a family of intermediate fibrous proteins expressed by many types of epithelial cells to varying degrees. The subset of cytokeratins expressed by epithelial cells depends on the type of epithelium. For example, cytokeratins 7, 8, 18, and 19 are all expressed by the normal monolayer epithelium of the kidney and remaining genitourinary tract, as well as the digestive and respiratory tracts. These cytokeratins in combination contribute to the structural integrity of epithelial cells. This combination represents both acidic (Type I) and basic (Type II) keratin families and is found to be abundantly expressed in kidney cells (Oosterwijk). et al. , J Histochem Cytochem, 38(3):385-392, 1990). Preferred cytokeratins for use in this specification are CK8, CK18, CK19 and combinations thereof.

[0364] Aquaporins are transport membrane proteins that allow the passage of water into and out of cells while blocking the passage of ions and other solutes. Thirteen aquaporins are described in this literature, six of which are found in the kidney (Nielsen et al., J Histochem Cytochem, 38(3):385-392, 2002). By imposing strict regulations to control water flow, aquaporin 2 results in a plasma membrane of renal collecting duct epithelial cells that is highly permeable to water, thus allowing water to flow in the direction of the osmotic gradient (Bedford et al. , J Am Soc Nephrol, 14(10):2581-2587, 2003; Takata et al. , Histochem Cell Biol, 130(2):197-209, 2008; Tamma et al. , Endocrinology, 148(3):1118-1130, 2007). Aquaporin 1 is a characteristic of the proximal tubule (Baer et al. , Cells Tissues Organs:184(1), 16-22, 2006; Nielsen et al. , 2002, supra ).

[0365] Curbilin is a transport membrane receptor protein. When it is co-located with the protein megarin, they together promote the internalization of curbilin-bound ligands, such as albumin. Curbilin is located within the epithelium of the intestine and kidney (Christensen, Am J Physiol Renal Physiol, 280(4):F562-573, 2001).

[0366] CXCR4 is a transport membrane protein that acts as a chemokine receptor for SDF1. Upon ligand binding, intracellular calcium levels increase, and MAPK1 / MAPK3 activation is enhanced. CXCR4 is naturally expressed in the kidney and plays an important role in renal development and tubular formation (Ueland et al. , Dev Dyn, 238(5):1083-1091, 2009).

[0367] Cadherins are calcium-dependent cell adhesion proteins. Cadherins are classified into four groups, where E-cadherins are found in epithelial tissues and are involved in regulating motility and proliferation. E-cadherins are transmembrane glycoproteins found to be located at the adhesion junctions of epithelial cells composing the distal tubules in the kidney (Prozialeck et al. , BMC Physiol, 4:10, 2004; Shen et al. , Mod Pathol, 18(7):933-940, 2005).

[0368] DBA (Dolichos biflorus agglutinin) is an α-N-acetylgalactosamine-binding lectin (cell-binding protein) that moves on the surface of the renal collecting duct structure, and is considered and used as a general marker for the development of the renal collecting duct and distal tubule (Michael et al. , J Anat 210(1):89-97, 2007; Lazzeri et al. , J Am Soc Nephrol 18 (12):3128-3138, 2007).

[0369] Differentiation cluster 31 (CD31; also known as the platelet-endothelial cell adhesion molecule, PECAM-1) is a cell adhesion protein expressed by selective populations of immune cells and endothelial cells. In endothelial cells, this protein is concentrated at cell boundaries (DeLisser, 1997). Differentiation cluster 146 (CD146) is involved in the cell adhesion and aggregation of endothelial cells at intracellular junctions associated with the actin cytoskeleton. Strongly expressed by vascular endothelium and smooth muscle, CD146 is currently used as a marker for the endothelial cell lineage (Malyszko et al. , J Clin Endocrinol Metab, 89(9):4620-4627, 2004), and is equivalent to CD31.

[0370] Gamma-glutamyl transpeptidase (GGT) is a metabolic enzyme that catalyzes the transport of the gamma-glutamyl moiety of glutathione to a recipient that can be an amino acid, peptide, or water. This enzyme also plays a crucial role in the synthesis and breakdown of glutathione and in the transport of amino acids across cell membranes. GGT is present in the cell membranes of many tissues, including the proximal tubular cells of the kidney (Horiuchi et al. , Eur J Biochem, 87(3):429-437, 1978; Pretlow et al. , J Histochem Cytochem, 35(4):483-487, 1987; Welbourne et al. , Am J Physiol, 277(4 Pt 2):F501-505, 1999). Table 12.1 It provides a list of specific types of kidney cells expressing these markers as detected by flow cytometry.

[0371] <Table 12.1>

[0372]

[0373] Regarding the phenotype of specific biomarkers Example 5 The SRC cells of were studied.

[0374] For immunophenotyping: Specific antibodies were added to 100 microliters of cell suspension, and the mixture was incubated in a dark room at 4°C for 30–45 minutes. After incubation, cells were washed with PBS and centrifuged to remove excess antibodies. Cells were resuspended in cell / microliter PBS and analyzed by flow cytometry. Flow cytometry analysis was performed using a FACSAria flow cytometer (Becton Dickinson) and FlowJo software (Treestar, Inc.). The antibodies used to characterize surface marker phenotypes are Tables 12.2 and 12.3It appears in. Isotype-specific primary antibody negative controls were used in all experiments. Appropriate isotype-matched controls were used to gate the negative population.

[0375] <Table 12.2>

[0376]

[0377] <Table 12.3>

[0378]

[0379]

[0380] Cell suspensions were generated from the initial tissue dissociation or trypsinization of adherent cultures and analyzed by flow cytometry to identify cellular components. The antibodies used were Table 12.2 and 12.3 As listed above. Isotype-specific primary antibody negative controls were used in all experiments. Labeled cells were analyzed using an ACSAria flow cell counter (Becton Dickinson) and FlowJo software (Treestar, Inc.). Appropriate isotype-matched controls were used to gate the negative population. After overnight incubation at 4°C, cells were pelleted, washed twice with Triton buffer (0.2% Triton X-100 in PBS), resuspended in 1 mL of DBPS containing the secondary antibody goat anti-mouse IgG2A conjugated with the fluorescent dye Alexa A647 (Invitrogen), and incubated for an additional 30 minutes. Subsequently, cells were washed for analysis using FACSAria and FlowJo software according to the manufacturer's instructions and resuspended in 1 mL of PBS. As a negative control, cells were incubated with the same fluorescent dye and a fitted isotype-matched monoclonal antibody.

[0381] Fig. 5(Phenotype distribution) shows the quantified expression of these markers in the SRC population plotted as the percentage values ​​of each phenotype in the population.

[0382] CK8 / 18 / 19 are the most consistently expressed kidney cell proteins detected across species. GGT1 and aquaporin-1 (AQP1) are consistently expressed, but their levels vary. Additionally, DBA, aquaporin 2 (AQP2), E-cadherin (CAD), CK7, and CXCR4 are observed at moderate levels, although they are more variable, while CD31 / 146 and curbilin have the lowest expression. Table 12.4 It provides the selected markers, the range and average percentage values ​​of the phenotypes in SRC, and the rationale for these selections.

[0383] <Table 12.4>

[0384]

[0385] SRC gene expression

[0386] The gene expression profiles of SRCs isolated from human kidney cell cultures by quantitative real-time polymerase chain reaction (qPCR) include those of aquaporin 2, E-cadherin, curbilin, VEGF, and CD31, which were also examined for protein production. Table 12.5 The genotype markers in [the text] represent the cell populations that can be expected to be found in renal cell cultures. NCAD, curbilin, and CYP2R1 are markers for tubular epithelial cells, and AQP2 and ECAD are markers for collecting ducts and distal tubules. Podocin and nephrine are markers for podocytes. VEGF and CD31 are endothelial markers. VEGF and EPO are oxygen-responsive genes associated with mRNA that exist in various different tissues and cell types.

[0387] The gene probes used were obtained from TaqMan. Passage 2 human kidney cells were harvested at 70–90% confluence. RNA was purified from the cells using Qiagen’s RNeasy Plus Mini Kit according to the protocol for the purification of total RNA from animal cells. Invitrogen’s SuperScript was used in accordance with the manufacturer’s instructions. ® cDNA was generated from a dose of RNA equivalent to 1.4 μg using the VILO™ cDNA synthesis kit. The mean qPCR data for the SRC population (n=3) Table 12.5 It appears in.

[0388] The results suggest the presence of a population of tubular epithelial cells, as evidenced by the relatively higher expression levels of NCAD, curbilin, and CYP2R1. Distal collecting duct tubules and distal tubular markers AQP2 and ECAD are relatively low, and even the endothelial marker, CD31, is lower ( Table 12.5 ).

[0389] <Table 12.5>

[0390]

[0391] Phenotypic and functional markers were selected based on the initial genotype assessment. VEGF gene expression levels were high and aquaporin 2 gene expression levels were low, which is consistent with the protein analysis data ( Tables 12.4 and 12.6 ).

[0392] SRC enzyme activity

[0393] The existence of live cells and SRC function were proven through the metabolism of PrestoBlue and the production of VEGF and KIM-1.

[0394] SRC actively secretes proteins that can be detected through the analysis of the conditioning medium. Cell function is evaluated by cell activity, metabolizes PrestoBlue, and secretes VEGF (vascular endothelial growth factor) and KIM-1 (kidney injury molecule-1).

[0395] Their ability to metabolize PrestoBlue allows for the monitoring of viable functional cells in NKA. PrestoBlue is a modified resazurin-based assay reagent that is a cell-permeable, non-fluorescent blue dye. Upon influx into viable cells sufficient for surging, the dye is reduced to a bright red fluorescent dye, which can be measured as fluorescence or absorbance, through natural cellular processes involving dehydrogenase enzymes.

[0396] The biomolecules VEGF and KIM-1 represent the selection of molecules from those proposed as nonclinical biomarkers for sensitive and specific analyses of renal injury and function (Sistare, 2010; Warnock, 2010). In vivo, both of these markers indicate tubular function, injury, and / or repair, and in vitro, they are recognized as characteristics of tubular epithelial cell cultures. KIM-1 is an extracellular protein anchored to the membrane of renal proximal tubular cells that serves to recognize and phagocytose apoptotic cells shed during injury and cellular metabolic turnover. Innately expressed by renal cells, VEGF is a key angiogenic and pro-survival factor that promotes cell division, migration, endothelial cell survival, and angiogenesis. SRC innately expresses VEGF mRNA and ( Table 12.5 Actively producing ) proteins ( Table 12.6 These proteins were characterized as being ) and can be detected in kidney cells and culture media exposed to SRC. Table 12.6 Figure [] represents the quantification of VEGF and KIM-1 present in the conditioning medium from kidney cells and SRC cultures. Kidney cells were cultured until near confluence. The conditioning medium from kidney cell cultures and SRC exposed overnight was tested for VEGF and KIM-1.

[0397] <Table 12.6>

[0398]

[0399] The cellular function of the pre-preparation, SRC, was also evaluated by measuring the activity of two specific enzymes found in the proximal tubules of the kidney: GGT (γ-glutamyl transpeptidase) and LAP (leucine aminopeptidase) (Chung, 1982, J Cell Biol 95(1):118-126). The method for measuring the activity of these enzymes in cells utilizes enzyme-specific substrates in solution that are cleaved and release chromogenic products when added to cells expressing the active enzyme (Nachlas, 1960 J Biophys Biochem Cytol 7:261-264; Tate, 1974 Proc Natl Acad Sci USA 71(9):3329-3333). The absorbance of the cell-exposed solution is measured, and said absorbance is related to the amount of cleaving products originating from the active enzyme. The substrate utilized for GGT is L-glutamic acid γ-p-nitroanalide hydrochloride, and the substrate utilized for LAP is L-leucine p-nitroanalide. Fig. 6 It shows LAP and GGT activity in 6 SRC samples generated from human donors (BP1-BP4).

[0400] Summary of SRC Characterization

[0401] Cell morphology during cell expansion was monitored by comparing culture observations using images from the Image Library. Cell growth kinetics were monitored at each cell passage. Cell growth is expected to be patient-specific. SRC number and variability were monitored by trypan blue dye exclusion and / or PrestoBlue metabolism. SRCs are characterized by the phenotypic expression of CK18 and GGT1. Additionally, AQP2 expression can be monitored. PrestoBlue metabolism and the production of VEGF and KIM-1 are used as markers for the presence of viable and functional SRCs. Furthermore, SRC function can be elucidated through the measurement of enzymatic activity toward LAP and GGT and gene expression profiling.

[0402] Example 13

[0403] Biomaterial manufacturing

[0404] The biomaterial used in NKA (gelatin solution) is characterized through two main parameters:

[0405] density - Measure the concentration of the gelatin solution using the absorbance at 280 nm with a spectrophotometer. Determine the gelatin concentration from the absorbance versus concentration calibration curve.

[0406] Inversion test - The inversion test provides a visible evaluation of the gelatin solution's ability to form and maintain a gel at a temperature of 2-8°C and its ability to liquefy the gel at room temperature.

[0407] Explanation of the characteristics of other biomaterials

[0408] The biomaterials used in NKA can be further characterized for rheological properties and viscosity. Rheological and viscosity tests will be performed solely for verification purposes and are intended to be used for the extended characterization of biomaterials obtained from other vendors.

[0409] The rheological properties of biomaterials can be measured initially at 4°C and then at 25°C using a Couette cell-type flowmeter. The samples are equilibrated at each temperature for at least 30 minutes. The acceptable storage modulus (G'>10) at lower temperatures reflects the solution's ability to form and maintain a gel at NKA delivery and transport temperatures of 2–8°C. The acceptable loss modulus (G'<10) at higher temperatures reflects the gel's ability to liquefy at room temperature, as required for NKA delivery and transplantation.

[0410] The viscosity of biomaterials is measured using conical and plate viscometers at a shear rate of 200-300 s⁻¹ and 37°C. Solutions with viscosities in the range of 1.05-1.35 cP can be effectively delivered through an 18-27 Gauge needle.

[0411] In the preparation of NKA formulations, a gelatin solution is prepared by dissolving gelatin in DPBS to a specified concentration (0.88% ± 0.12% w / v). Figure 2D ). The gelatin solution was filtered through a 0.1 μm filter, sterilized, and divided into equal portions within tubes. Samples were taken for release of the gelatin solution prior to freezing or preparation of NKA. As a bulk material prepared for preparation, the gelled hydrogel is stored refrigerated or frozen ( Figure 2D ).

[0412] Example 14

[0413] NKA preparations

[0414] By excluding trypan blue dye Example 5The number of washed SRCs was counted. The gelatin solution was removed from cold storage and liquefied by preheating to 26–30°C. The volume of SRC suspension containing the required number of cells was centrifuged and resuspended in the liquefied gelatin solution for the final washing step. This suspension was centrifuged, and the SRC pellet was resuspended in a sufficient amount of gelatin solution to 100x10 in the prepared NKA. 6 A resulting SRC concentration of 1 cell / mL was achieved ( Figure 2D ).

[0415] NKA is present in a sterile, single-use 10 mL syringe. 100x10 6 Concentration of NKA at 3.0 x 10⁻¹⁰ SRC / mL and 3.0 x 10⁻¹⁰ 6 The final volume was calculated from the target dose of dog SRC / g kidney weight (estimated by MRI).

[0416] Example 15

[0417] NKA filling and gelation

[0418] NKA products were aseptically filled into syringes of NKA packages in the BSC for tissue processing and cell culture operations ( Figure 2D During the filling process, dynamic air sampling was performed, including viable and non-viable sampling.

[0419] The prepared NKA was placed in a 50 mL sterile centrifuge tube. A sterile cannula was attached to a 10 mL delivery syringe. NKA was manually withdrawn from the 50 mL tube into the delivery syringe through the cannula. The cannula was removed, and the delivery syringe was connected to a Luer-lock fitting the end of the NKA package tube. NKA was transferred to the syringe of the NKA package by pressing the plunger on the delivery syringe. At least 8.5 mL of product was transferred to the syringe of the NKA package. The syringe was inverted, and trapped air was removed by slowly pressing the plunger. After filling was complete, the tubing in the NKA package was sealed with RF sealant. The remaining product in the delivery syringe was returned to the 50 mL tube. A quality control (release test) sample was taken from the 50 mL tube. The NKA package was rotated for at least 2 hours to maintain the cells in the suspension and simultaneously cooled to 2-8°C to form the final gelled NKA ( Figure 2D ).

[0420] Rapid cooling was required for the gelation to occur so that the cells would not precipitate in the gelatin solution. Since it was kept refrigerated, the temperature of the gelatin solution in the syringe was monitored. A rapid decrease in temperature was observed. After 1 hour, the temperature dropped from a final temperature of 4.4℃ to within 0.3℃.

[0421] Cooling of the gelatin solution initiates the gelation process, but a finite amount over time is required to stabilize the formed gel; therefore, SRC will remain suspended in the gel during storage. Syringes containing the prepared NKA were spun overnight or for 1.25 hours and then held vertically overnight. Subsequently, the inclusions were removed, and cell concentrations were measured in four different sections of the product. The analysis showed no difference among the four sections, indicating that no measurable cell precipitation occurs after NKA is spun at a cold temperature for at least 1.25 hours (data not shown).

[0422] Example 16

[0423] NKA Packaging and Shipping

[0424] The NKA was packed in an NKA delivery box along with appropriate documentation. The delivery box was designed to maintain a temperature in the range of 2–8°C during transport to the hospital. The cooled (gelled) preparation has a shelf life of 3 days.

[0425] Temperature loggers were included in the NKA packages to monitor temperature during delivery. The number of NKA batches was verified against unique patient IDs recorded in the delivery / receipt logbook by the quality group. The NKA boxes were delivered to the hospital via a courier company or a similar secure transport agency.

[0426] Example 17

[0427] Transplantation of NKA (SRC cell population)

[0428] This example demonstrates the regenerative properties of selected heterogeneous human kidney cells.

[0429] NKA delivery system

[0430] The NKA delivery system consisted of cannulas (needles) and syringes suitable for cell delivery. Different vendors use the terms cannula or needle to describe cell delivery products. For this description, the terms cannula and needle are used interchangeably. The proposed clinical trial utilized the same delivery system (cannulas and syringes) used in animal studies adapted to human size and dissection. Laparoscopic surgical procedures were used.

[0431] The main component of the NKA delivery system was the cannula. A cannula suitable for NKA was used.

[0432] NKA transplant

[0433] In preparation for transplantation, the NKA was preheated to room temperature immediately before injection into the kidney to liquefy the product.

[0434] NKA was the target for injection into the renal cortex via a needle or cannula and syringe suitable for cell delivery. The use of a pointed needle (cannula) to penetrate the renal capsule allowed the introduction of the delivery needle / cannula into the renal cortex. A syringe containing NKA was attached to the delivery needle, and NKA was injected into multiple sites of the renal cortex. Fig. 7 The illustration in [location] explains the concept of injecting NKA into the kidney using a needle suitable for cell delivery and distribution into solid organs.

[0435] NKA was delivered directly into the renal cortex. NKA delivery in the patient was performed using standardized laparoscopic surgery.

[0436] It is understood that the embodiments and specific examples described herein are for illustrative purposes only, and that various modifications or variations will be presented to those skilled in the art in light of the teachings provided, and shall be contained within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications listed herein are incorporated herein by reference in their entirety for all purposes.

Claims

Claim 1 A method for preparing a pharmaceutical composition comprising a gelatin hydrogel biomaterial and selected kidney cells (SRC), comprising: i) a step of preparing a liquefied gelatin solution, wherein the liquefied gelatin solution comprises a temperature of about 26°C to about 30°C; ii) a step of suspending the SRC in the liquefied gelatin solution; and iii) a step of rapidly cooling an SRC suspended in a liquefied gelatin solution, wherein the rapid cooling comprises lowering the temperature of the liquefied gelatin solution containing the SRC to about 2°C to about 8°C within about 2 hours, and wherein the rapid cooling comprises forming a pharmaceutical composition by gelling the liquefied gelatin solution containing the SRC into a gelatin hydrogel biomaterial containing the SRC, wherein the SRC comprises (i) cells expressing gamma-glutamyl transpeptidase (GGT-1), (ii) cells expressing cytokeratin (CK), and (iii) cells secreting vascular endothelial growth factor (VEGF) and kidney injury molecule (KIM)-1, wherein more than 4.5% of the SRC expresses GGT-1 and more than 80% of the SRC expresses CK. Claim 2 A method according to claim 1, wherein rapid cooling causes the temperature to drop within approximately 1.25 hours. Claim 3 A method according to paragraph 2, wherein rapid cooling involves lowering the temperature to within 0.3°C of 4.4°C within one hour. Claim 4 In claim 1, approximately 100x10 SRC in the liquefied gelatin solution 6 A method in which a gelatin solution is suspended at a concentration of SRC / mL. Claim 5 In paragraph 2, approximately 100x10 SRC in the liquefied gelatin solution 6 A method in which a gelatin solution is suspended at a concentration of SRC / mL. Claim 6 In paragraph 3, approximately 100x10 SRC in the liquefied gelatin solution 6 A method in which a gelatin solution is suspended at a concentration of SRC / mL. Claim 7 A method according to claim 1, wherein the liquefied gelatin solution comprises gelatin dissolved in Dulbecco phosphate buffered saline (DPBS), and said gelatin has a concentration of 0.88% ± 0.12% w / v. Claim 8 A method according to claim 2, wherein the liquefied gelatin solution comprises gelatin dissolved in DPBS, and said gelatin has a concentration of 0.88% ± 0.12% w / v. Claim 9 A method according to claim 4, wherein the liquefied gelatin solution comprises gelatin dissolved in DPBS, and said gelatin has a concentration of 0.88% ± 0.12% w / v. Claim 10 A method according to claim 1, further comprising a characterization step of SRC prior to the manufacturing step, wherein the characterization step comprises determining (i) whether more than 4.5% of the SRC expresses GGT-1, (ii) whether more than 80% of the SRC expresses CK, and (iii) whether the cells of the SRC secrete VEGF and KIM-1. Claim 11 A method according to claim 4, further comprising a characterization step of SRC prior to the manufacturing step, wherein the characterization step comprises determining (i) whether more than 4.5% of the SRC expresses GGT-1, (ii) whether more than 80% of the SRC expresses CK, and (iii) whether the cells of the SRC secrete VEGF and KIM-1. Claim 12 A method according to claim 7, further comprising a characterization step of SRC prior to the manufacturing step, wherein the characterization step comprises determining (i) whether more than 4.5% of the SRC expresses GGT-1, (ii) whether more than 80% of the SRC expresses CK, and (iii) whether the cells of the SRC secrete VEGF and KIM-1. Claim 13 A method according to claim 1, wherein the liquefied gelatin solution comprises gelatin dissolved in PBS.

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